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Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.

To overcome the problem of in-stent restenosis, the concept of local delivery of antiproliferative or immunosuppressive drugs has been introduced into interventional cardiology. Local drug delivery can be achieved by drug-eluting stents coated with polymer surfaces used for controlled drug release. However, several polymer coatings have shown an induction of inflammatory response and increased neointima formation. In the present study, the effect of a new inorganic ceramic nanoporous aluminum oxide (Al(2)O(3)) coating on neointima proliferation and its suitability as a carrier for the immunosuppressive drug tacrolimus have been investigated. 316 L stainless steel coronary stents were coated with a 500 nm thin nanoporous aluminum oxide layer. This ceramic nanolayer was used as a carrier for tacrolimus. Bare stents (n = 6), ceramic coated stents (n = 6), and ceramic coated stents loaded with 60 (n = 7) and 120 mug (n = 6) tacrolimus were implanted in the common carotid artery of New Zealand rabbits. The ceramic coating caused no significant reduction of neointimal thickness after 28 days. Loading the ceramic stents with tacrolimus led to a significant reduction of neointima thickness by 52% for 60 mug (P = 0.047) and 56% for 120 mug (P = 0.036) as compared to the bare stents. The ceramic coating alone as well as in combination with tacrolimus led to a reduced infiltration of lymphocytes and macrophages in the intima in response to stent implantation. Ceramic coating of coronary stents with a nanoporous layer of aluminum oxide in combination with tacrolimus resulted in a significant reduction in neointima formation and inflammatory response. The synergistic effects of the ceramic coating and tacrolimus suggest that this new approach may have a high potential to translate into clinical benefit., (Copyright 2003 Wiley-Liss, Inc.)

Arterial interstitial cells of Cajal (ICC)-like cells (AIL cells) with a multipolar, irregular, elongated shape and with numerous thin (often less than 1 microm), sometimes branching, processes with lengths up to approximately 60 microm were isolated enzymatically from 1st to 7th order branches of guinea-pig mesenteric artery. Some of the processes of AIL cells were growing (average speed approximately 0.15 microm min-1) and their growth was blocked by 10 microM latrunculin B, an inhibitor of actin polymerisation. Staining with BODIPY phalloidin, a fluorescent dye selective for F-actin, showed the presence of F-actin in the processes of AIL cells. Voltage clamp of single AIL cells revealed an inward current that was four times more dense than in myocytes and was abolished by 10 microM nicardipine, and an outward current carried exclusively by potassium ions that was reduced by 1 mM 4-aminopyridine and/or 100 nM iberiotoxin but unaffected by 10 nM dendrotoxin-K. Imaging of intracellular ionised calcium with fluo-4 using a laser scanning confocal microscope showed local or global calcium transients lasting several seconds in approximately 28 % of AIL cells. When membrane current was recorded simultaneously, the calcium transients were found to correspond to long-lasting transient outward currents, which occurred at potentials positive to -40 mV. Unlike myocytes, AIL cells did not contract in response to 1 mM caffeine or 5 microM noradrenaline, although they responded with a [Ca2+]i increase. The segments of intact arteries did not stain for c-kit, a marker of ICCs. Single AIL cells stained positive for vimentin, desmin and smooth muscle myosin. The presence of ICC-like cells is demonstrated for the first time in the media of resistance arteries.

There is an abundance of ultrastructural data in the literature on vascular, visceral, and other smooth muscles; such data on airway smooth muscle, however, are conspicuously missing. Here we present a series of electron micrographs depicting contractile and cytoskeletal elements as well as organelles in porcine trachealis. Myosin thick filaments are present in the relaxed muscle; thick filament density increases substantially when the muscle is activated. Actin thin filaments are present in large excess over the thick filaments; the thin/thick filament ratio is about 31/1 in the relaxed state; this ratio is reduced to about 22/1 when the muscle is activated. The sarcoplasmic reticulum is often found associated with caveolae and mitochondria. Cells within a bundle are well connected by intermediate and gap junctions. The results demonstrate that quantitative morphological analysis of ultrastructure of airway smooth muscle fixed under different functional states is possible and will be essential in elucidating the structural basis of adaptation and contraction of the muscle.

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.

During the atheroma plaque formation, smooth muscle cells (SMC) have to change their differentiated phenotype in order to proliferate, migrate and synthesize collagen. These phenotypic changes are stimulated by insulin and IGF-1, and we have studied the effect of L-type calcium channel blockade produced by diltiazem on such changes. Mitotic activity was measured using bromodeoxyuridine DNA incorporation, the migration capability as chemotaxis index in a Boyden chamber, and cytoskeleton changes related to SMC movement in immunofluorescence studies. Diltiazem (10(-7)-10(-6) M) reduced insulin-induced mitotic activity in cultured human vascular SMC more effectively than in IGF-1-induced mitotic activity, but at 10(-5) M, the inhibitory effects were similar. Diltiazem also showed a clear inhibition of migration ability, both under basal conditions (p < 0.05) and after addition of insulin (p = 0.0001) and IGF-1 (p < 0.0001). Finally, diltiazem inhibited membrane ruffling induced both by insulin and IGF-1 in a similar manner, and similar results were obtained with SMC from rat aorta. We conclude that substances blocking the L-type calcium channels such as diltiazem, could inhibit those processes which in vivo lead SMC to form the atheroma plaque.

Ultrastructural study of the small intestine in suckling rabbits with experimental cholera revealed involvement of the inner and outer smooth muscle layers into the pathological process. Smooth muscle cells were characterized by vacuolar and fatty degeneration and focal colliquative necrosis. Apoptosis played little role in gastrointestinal motility disturbances. The presence of considerable amounts of fluid in intestinal loops reflects peristaltic dysfunction due to generalized damage to smooth muscle cells.

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors., (Copyright 2003 Wiley-Liss, Inc.)

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% +/- 5.7 (SE) and 76.0% +/- 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% +/- 6.7, 34.6% +/- 3.4, and 35.6% +/- 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length.

The histogenesis of the aggressive angiomyxoma of the vulvo-vaginal region was studied. Four cases of aggressive angiomyxoma were examined by using light microscopy, electron microscopy, and immunohistochemistry. Myofibroblastic from smooth muscle cell differentiation was distinguished by paying close attention to the structures of the neoplastic cell membrane. Aggressive angiomyxomas exhibit subtle features of smooth muscle differentiation, suggesting that the neoplastic cells differentiate from a multipotent perivascular cell.

Inflammatory myofibroblastic tumor (IMT), synonymously referred to as inflammatory pseudotumor, is a distinctive mesenchymal lesion composed of spindle cells displaying morphological features of myofibroblasts admixed with considerable numbers of inflammatory cells. Recent genetic and molecular studies have shown that a subset of IMT is characterized by the expression of altered anaplastic lymphoma kinase (ALK) protein mostly resulting from rearrangements of the ALK gene such as TPM3-ALK, TPM4-ALK and CLTC-ALK fusion genes. We analyzed the ALK status in nine cases of IMT arising in various anatomical locations. Six cases showed immunohistochemical expression of the ALK protein, and two ALK-positive lesions examined by reverse transcription-polymerase chain reaction and a subsequent sequencing analysis harbored the TPM4-ALK fusion gene. Of note, the majority of ALK-positive tumor cells in four of the six lesions lacked the coexpression of myogenic markers including alpha-smooth muscle actin, a cytoskeletal protein indicating myofibroblastic differentiation, whereas a substantial number of tumor cells in the remaining two cases coexpressed ALK and alpha-smooth muscle actin and/or desmin. In an ultrastructural study of the lesion with predominant ALK-positive/actin-negative cells, spindle cells failed to demonstrate features of myofibroblasts such as intracytoplasmic bundles of thin filaments and dense bodies. The current findings suggest that ALK-positive cells in IMT are not always myofibroblastic but might be immature primitive mesenchymal cells.

Pompe's disease (glycogen storage disease type II) is an autosomal recessive myopathy caused by lysosomal alpha-glucosidase deficiency. Enzyme replacement therapy (ERT) is currently under development for this disease. We evaluated the morphological changes in muscle tissue of four children with infantile Pompe's disease who received recombinant human alpha-glucosidase from rabbit milk for 72 weeks. The patients were 2.5-8 months of age at entry. Prior to treatment, all patients showed lysosomal glycogen storage in skeletal and smooth muscle cells, vascular endothelium, Schwann cells, and perineurium. The first response to treatment was noticed in vascular endothelium and in peripheral nerves after 12 weeks of treatment at an enzyme dose of 15-20 mg/kg. Increasing the dose to 40 mg/kg led, after 72 weeks of treatment, to a reduction of glycogen storage and substantial improvement of muscle architecture in the least affected patient. Not all patients responded equally well, possibly due to differences in degree of glycogen storage and concomitant muscle pathology at the start of treatment. We conclude that intravenous administration of recombinant human alpha-glucosidase from rabbit milk can improve muscle morphology in classic infantile Pompe's disease when treatment is started before irreversible damage has occurred.

Introduction: The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery., Methods and Results: In the cell-attached configuration, membrane stretch with negative pressure induced two types of stretch-activated (SA) ion channels: a nonselective cation channel and a large-conductance Ca2+-activated K+ (BK(Ca)) channel. The single-channel conductances of SA cation and BK(Ca) channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on-cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BK(Ca) channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BK(Ca) channel activity was greatly enhanced, but the activity of SA cation channels disappeared., Conclusion: This study is the first to demonstrate that the behavior of SA cation and BK(Ca) channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension.

4-Hydroxy-2-nonenal (HNE) has been suggested to contribute to the pathogenesis of atherosclerosis. One of the major metabolic transformation pathways of HNE involves conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST). In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat aortic smooth muscle A10 cells. Incubation of A10 cells with D3T resulted in a marked concentration- dependent induction of both GSH and GST. The induction of cellular GST by D3T also exhibited a time-dependent response. Pretreatment of A10 cells with D3T led to a dramatic decrease of HNE-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay and scanning electron microscopy. Incubation of A10 cells with HNE for 0.5 h and 1 h resulted in a significant depletion of cellular GSH, which preceded the decrease of cell viability. To further demonstrate the involvement of GSH and GST in protecting against HNE-induced cytotoxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of GSH by BSO or inhibition of GST by sulfasalazine caused great potentiation of HNE-mediated cytotoxicity. Moreover, cotreatment of A10 cells with BSO was found to completely block the D3T-mediated GSH induction and to largely reverse the cytoprotective effects of D3T on HNE-induced toxicity. Taken together, this study demonstrates that D3T can induce both GSH and GST in aortic smooth muscle cells, and that the D3T-augmented cellular defenses afford a marked protection against HNE-induced vascular cell injury.

Expression of the receptor tyrosine kinase KIT on cells referred to as interstitial cells of Cajal (ICC) has been instrumental during the past decade in the tremendous interest in cells in the interstitium of the smooth muscle layers of the digestive tract. ICC generate the pacemaker component (electrical slow waves of depolarization) of the smooth musculature and are involved in neurotransmission. By integration of ICC functions, substantial progress has been made in our understanding of the neuromuscular control of gastrointestinal motility, opening novel therapeutic perspectives. In this article, the ultrastructure and light microscopic morphology, as well as the functions and the development of ICC and of neighboring fibroblast-like cells (FLC), are critically reviewed. Directions for future research are considered and a unifying concept of mesenchymal cells, either KIT positive (the "ICC") or KIT negative "non-Cajal" (including the FLC and possibly also other cell types) cell types in the interstitium of the smooth musculature of the gastrointestinal tract, is proposed. Furthermore, evidence is accumulating to suggest that, as postulated by Santiago Ramon y Cajal, the concept of interstitial cells is not likely to be restricted to the gastrointestinal musculature.

Objective: Lower urinary tract symptoms (LUTS) in elderly males are not solely caused by bladder outlet obstruction (BOO) and may be at least partly attributable to detrusor dysfunction. Urodynamically, patients may show instability, hypocontractility, BOO or combinations of these findings. These findings have been related to specific ultrastructural changes in detrusor smooth muscle cells; however, this relationship is controversial. The aim of this study was to correlate ultrastructural findings in patients with BOO with urodynamic parameters., Material and Methods: In 25 men with BOO verified by means of a full urodynamic evaluation, including a pressure-flow study, a detrusor biopsy was obtained. Six men without BOO served as controls. Biopsies for electron microscopy were analysed semiquantitatively and morphometrically to determine the presence of muscle cell hypertrophy, variation in intercellular distances, occurrence of abnormal cell junctions and configurations and intracellular changes., Results: The only parameter which was found to relate to the degree of obstruction in BOO was an increase in intra- and interfascicular elastin, all other correlations not reaching significance., Conclusion: This study does not confirm a specific relationship between ultrastructural detrusor smooth muscle features and various types of BOO. Therefore ultrastructural investigation of detrusor smooth muscle cells cannot replace urodynamic evaluation in the classification of LUTS.

Background: The resistance to stretch and the elasticity of the vein wall depend on the collagen and elastic fibers, respectively. Contradicting evidence exists, however, on the connective tissue concentration in varicose veins., Methods: A prospective, comparative study was conducted at Asir Central Hospital and the College of Medicine in Abha, Saudi Arabia. Twenty-three vein specimens collected from both the proximal thigh long saphenous vein (LSV) and the distal calf blowouts in 10 primary varicose vein patients and from the normal, proximal thigh LSV in 3 young vascular trauma patients were examined. Paraffin sections stained with hematoxylin and eosin, Verhoeff von Gieson (VVG) and Masson's Trichrome stains were examined under the light microscope. Ultra thin sections were examined under the transmission electron microscope (TEM)., Results: Compared with the normal control LSV, varicose vein sections showed increased diameter of the lumen and hypertrophy of the wall, mainly of the intima, due to increased amounts of collagen fibers. This marked fibrous infiltration disrupted the normal palisade arrangement of the intimal and the regular sheet-like arrangement of the medial smooth muscle cells. Collagen fibers also lost their normal pattern and showed abnormal forms. Elastic fibers lost their regular laminar arrangement and formed clumps or scattered fragments., Conclusions: Varicose veins showed increased collagenosis and distortion of the elastic fibers. The presence of abnormal collagen to elastin ratio and the loss of the regular collagen/elastic lattice of the vein wall may play a major role in the pathogenesis of varicose veins.

To elucidate the cellular mechanism underlying the growth of the peritoneal cover of the gut sinus and the heart in the polychaete Arenicola marina, cellular organization of these structures and proliferative potential of their cells were investigated using electron microscopy and electron microscopic autoradiography. Arenicola has a pair of dorsolaterally situated hearts connected to the gut sinus via a short duct and composed of two muscular layers separated by a layer of the extracellular matrix (ECM). The peritoneal cover of the gut sinus and the outer muscular layer of the heart present a myoepithelial layer resting on the ECM. The inner muscular layer of the heart is composed of myofibril-containing cells lacking well-defined polarity in arrangement of organelles. However, their persistent connection to branches of the ECM and the adherens-like intercellular junctions allow for considering the inner layer a modified myoepithelium. In the peritoneal cover of the gut sinus and in both myoepithelial layers of the heart, noncontractile epithelial cells have been observed. As determined by thymidine labeling, these epithelial cells are capable of DNA synthesis, while myoepithelial cells are not. Some suggestions are made about the myogenic nature of the epithelial cells in the investigated structures of A. marina., (Copyright 2002 Wiley-Liss, Inc.)

Aim: To investigate the role of intracellular calcium in muscarinic currents increased by hypoosmotic membrane stretch in gastric antral circular myocytes of the guinea pig., Methods: The whole cell patch-clamp technique was used, and the myocytes were isolated by collagenase. Cells were swelled by hypoosmotic solution (200 Osmmol/kg)., Results: The hypoosmotic membrane stretch markedly increased carbachol-induced muscarinic currents (ICCh). The ICCh and the increase of ICCh were completely blocked by quinidine 3 micromol/L, a specific muscarinic current blocker. In external calcium-free solution hypoosmotic membrane stretch could not increase ICCh, but in the presence of nicadipine 5 micromol/L, a L-type calcium channel blocker or gadolinium chloride 100 nmol/L, a stretch-activated cation channel blocker, the ICCh was still increased by hypoosmotic membrane stretch. When both nicardipine and gadolinium chloride were added into external solution, ICCh were not increased by hypoosmotic membrane stretch any more. Ryanodine, a calcium-induced calcium release (CICR) agonist completely blocked hypoosmotic membrane stretch-induced increase of ICCh., Conclusion: Hypoosmotic membrane stretch increased ICCh and the increment was related to influx of external calcium and CICR.

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.

Aims: To develop a method to preserve lipids in formalin-fixed tissues for staining in paraffin sections, and to illustrate its use in lung and brain of a fat embolism case, and in examples of fatty liver and atheroma., Methods and Results: A saturated solution of linoleic acid in 70% ethylene glycol was prepared and tissues were exposed to this for 3 days at 56 degrees C. These tissues were treated with 2% chromic acid at 4 degrees C for 24 h followed by 24 h in 5% sodium bicarbonate, with appropriate rinsing between solutions. Paraffin sections of these tissues were stained with a lipid-soluble dye such as Oil Red O. Examples of fat embolism, fatty liver, and atheroma were shown photographically as illustrations of expected results., Conclusions: The demonstration of fat embolism with good quality tissue detail is made practical by the method, which is convenient and inexpensive. The method appears to be generally applicable to tissue lipids of various sorts, as exemplified by adipose tissue, fatty liver, and atheroma.

Complications in arteriovenous fistula (AVF) occur in up to 35% of renal failure patients on hemodialysis. The most frequent complication is thrombosis, usually from stenotic lesions in the venous outflow system. To study the pre-existing smooth muscle changes in the cephalic vein of these patients, we prospectively collected a total of 17 cephalic vein specimens from 3 normal controls and 14 renal failure patients undergoing primary AVF construction on the chosen limb. After preparation, ultrathin sections were stained with uranyl and lead acetate and were examined under the transmission electron microscope (TEM). Compared with the normal controls, abnormal fibrous infiltration of the intima and the media and varying degrees of smooth muscle degenerative changes were observed in all the cephalic vein sections of renal failure patients. Smooth muscle cells (SMCs) lost their normal fusiform shape and were widely separated by increased amount of irregularly disposed, extracellular collagen fibers. Other cellular abnormalities included irregular cell membrane, granular cytoplasm, Peri- and Paranuclear vacuoles and mega mitochondria. SMCs also showed morphological expression of phagocytosis of collagen and elastic fibers as a sign of remodeling of the vein wall. In conclusion, pre-existing wall and smooth muscle changes were observed in all the cephalic vein sections of renal failure patients, which may contribute to the later complications of AVFs.

Purpose: Numerous reports suggest that Class 1 interferons (IFNs), particularly IFN-gamma, inhibit migration and proliferation of different types of human cells. The objective of the present study was to determine the effect of Class I IFNs on viability and growth characteristics of human aortic endothelial cells (ECs), smooth muscle cells (SMC) and fibroblasts (FBs) in vitro., Methods: Stainless-steel (316-l) disks were coated with fibrin meshwork containing IFN-gamma or IFN-alpha. The discs and IFN embedded meshwork were incubated with human EC, SMC and FB, and then cultured, whereas control cells were seeded onto uncoated surfaces or plain fibrin meshwork. Concentrations of recombinant IFN varied from 5 to 20 ng/cm(2). Assessment of effect on cell viability, growth and attachment was performed utilizing Alamar Blue (AB) assay. Cell morphology was assessed by scanning electron microscopy (SEM)., Results: We have now shown inhibitory capacity of IFN-gamma on all three types of unstimulated cells. The growth-inhibitory effect was maximal with SMC, while it was minimal with FB and EC. IFN-gamma abrogated mitogenic responses of SMC but not EC and partially FB to VEGF and FGF stimulation. IFN-alpha was able to inhibit EC growth and, to a lesser extent, FB, and did influence growth rates of SMC. Biochemical analysis of lactate dehydrogenase activity suggested that IFN was not toxic to vascular cells. We also measured the expression of cell adhesive molecules: P- and E-selections, PECAM and ICAM-1. These molecules were upregulated by IFN in EC. Media derived from quiescent human SMC displayed low immunoreactive elastase activity, while conditional media after IFN-gamma treatment but not IFN-alpha treatment had approximately a threefold greater activity., Conclusion: These data suggest that IFN-gamma significantly inhibits SMC growth in the absence of significant endothelial toxicity and is dose-dependent; however, animal experiments are needed to further explore the antirestenotic effects of IFNs.

The object of this study was to develop a compliant hybrid vascular graft minimally supported by an elastomeric scaffold for arterial replacement. The hybrid vascular grafts designed were composed of three layers: an inner surface lined with endothelial cells (ECs); a hybrid medial tissue composed of a collagenous gel embedded with smooth muscle cells (SMCs); and an outer layer made of a laser-processed micropored segmented polyurethane (SPU) film with the circular pore size (diameter 150 microm) but different film thickness (50-200 microm) and different pore-to-pore distances (1 or 4 mm). The approximate dimensions of the hybrid vascular graft without the SPU film were as follows: inner diameter, 5 mm; length, 5 cm; thickness, 50 microm. The intraluminal pressure-external diameter relationship was measured by infusion of a phosphate buffer solution into the hybrid vascular graft. Canine carotid arteries and commercially available ePTFE grafts served as controls. Decrease in the thickness of the SPU film and increase in the pore density of the SPU film increased the pressure-dependent distensibility of the hybrid vascular grafts. The thinner the film and higher the pore density, the more compliant was the hybrid graft. The pressure-induced distensibility of the designed hybrid graft was found to be close to that of native carotid arteries.

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919-3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg2+. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg(2+)-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments.

Peculiarities of terminal lymphangion wall in humans and dogs were studied in its different parts using various morphological methods. Main results were obtained with the use of a total preparation method after A.V. Borisov. Heterogenous distribution of myocytes in different regions of lymphangion (muscular cuff, valve sinus wall) was noted. In the terminal lymphangion myocytes from three anatomically and topographically different muscles: cuff muscle, tensor muscle of the distant valve, tensor muscle of the proximal valve (thoracic duct orifice valve). Some features of terminal lymphangion construction similar to those seen in other lymphangions as well as certain specific characteristics and species peculiarities, are described.