Long-term structural alterations to endothelial cells in vein-to-artery grafts: a quantitative electron microscopic study

The intracellular structure of endothelium lining vein-to-artery grafts in rats was analysed, using transmission electron microscopy and morphometry, to determine the ultrastructural adaptations of endothelial cells in this altered vascular environment. Autogenous 4-mm sections of iliolumbar veins were inserted microsurgically into the left common iliac arteries of 16 male Wistar rats. At 3, 6, 26 and 52 weeks the cytoplasmic-vesicular, mitochondrial and rough endoplasmic reticular contents of endothelial cells lining the grafts, the opposite iliac arteries and the remaining ilio-lumbar veins were analysed morphometrically. There was a significant increase in the amount of all these cytoplasmic structures in endothelial cells at 3, 6 and 26 weeks; at 52 weeks there was also a significant increase in the volumes of mitochondria and cytoplasmic vesicles, but not in rough endoplasmic reticulum. It was concluded that the ultrastructure of endothelial cells lining these grafts is changed chronically after graft insertion, and we propose that this may be attributable to altered haemodynamic stresses within the graft.     

Vascular tissue adaptations in end-to-end autologous arterial grafts in rats: a morphometric analysis

Autologous vein grafts are employed extensively to bypass stenoses in the arterial circulation. More recently arterial segments have been used for such bypass surgery. In this study the adaptation of regenerating vascular tissues in experimental autologous artery grafts (4 mm long and 1 mm in diameter) in 20 adult male Wistar rats was analysed. At 1, 2, 4, 8 and 16 wk after insertion, 4 grafts per time interval were removed, processed for high resolution light microscopy and the thicknesses of the media and neointima, as well as the area fractions of smooth muscle cells, were analysed morphometrically. All grafts were reendothelialised by 2 wk. Neointimal hyperplasia (a subendothelial layer of smooth muscle cells) developed in all grafts and reached its maximal thickness (40.4±4.7 μm) at 2 wk. The area fraction of smooth muscle cells in the neointima of the artery grafts did not change significantly at any time from 2 to 16 wk. The media underlying the neointima of the artery grafts remained relatively constant throughout the 16 wk duration of the experiment. Whilst the total wall thickness of the grafts reduced significantly between 2 and 4 wk after insertion, at all times the grafts were thicker than the host artery.     

Phase-plane analysis of action potentials in uterine smooth muscle

Summary Action potentials were recorded by micro-electrode from narrow strips of pregnant rat uterus in vitro. The phase-plane display (V vsdV/dt) of selected action potentials was analysed by the method of Jenerick (1964) to yield the ionic current. From this membrane current data, various parameters of the action potential were calculated. In comparison to skeletal muscle action potentials, the ionic currents were 30–100 times smaller in the uterus action potential. Epinephrine hyperpolarized the resting potentials and suppressed spontaneous activity, but did not cause any significant changes in the stimulated action potential. The after-potential may have been affected by epinephrine, preventing repetitive firing, but the data were inconclusive. The phase-plane analysis results were similar to the results of the double sucrose gap voltage clamp method on the same tissue (Kao and McCullough, 1975).A preliminary report of this work has been published (Specht and Kao, 1973)     

Presence of a smooth muscle system in aortic valve leaflets

Summary The location and the spatial arrangement of smooth muscle cells in aortic valves have been assessed by a systematic analysis of serial semithin sections of plastic embedded porcine and human aortic leaflets, combined with an electron microscope study.The investigation showed that smooth muscle cells, either single and arranged in thin bundles, and other cell types such as myofibroblasts are constantly present in the aortic valve leaflets. In addition, it was possible to devise a model of the three dimensional, specific organization of the smooth muscle bundles which can be interpreted as an intrinsic muscle system of the leaflets. As the muscular elements might play an active role in the normal functioning of the valve, their presence should be taken into account in designing (bio) prosthetic leaflets and in the evaluation of valve pathology.This work was supported by grant CT76 01159904 from CNR (Rome)     

移植动脉新生内膜平滑肌细胞来源于受体骨髓细胞的实验依据

目的： 研究大鼠移植动脉新生内膜平滑肌细胞Sry基因表达，探讨移植物动脉硬化新生内膜平滑肌细胞的来源。方法: 将雄性Wistar大鼠骨髓细胞移植到雌性大鼠体内，制备嵌合体大鼠；4周后建立大鼠腹主动脉移植慢性排斥反应模型，分为4组：雌性同系移植组、雌性异系移植组、雄性异系移植组、嵌合体异系移植组。移植术后8周，取移植动脉组织标本，进行病理组织学观察与免疫组化染色，分析移植动脉新生内膜增生及新生内膜细胞α-SMA的表达；采用显微切割技术收集新生内膜平滑肌细胞，用PCR扩增方法检测细胞Sry基因表达，分析新生内膜平滑肌细胞与受体骨髓细胞的关系。结果: 异系移植组移植动脉新生内膜中α-SMA表达阳性平滑肌细胞大量增生，致动脉内膜显著增厚，动脉壁新生内膜面积及新生内膜/中膜面积比均显著高于同系移植组(P<0.01)。PCR分析显示，嵌合体异系移植组和雄性异系移植组一致，在约225 bp处均有1条特异性扩增条带，而雌性异系移植组则无相应的核酸扩增条带。结论: 受体骨髓细胞作为移植物血管新生内膜平滑肌细胞的来源，参与移植物动脉硬化发生和发展。     

Complex multilocular cystic lesion of rete testis,accompanied by smooth muscle hyperplasia,mimicking intratesticular Leydig cell neoplasm

Non-neoplastic smooth muscle cell proliferation occurs under a variety of circumstances in many body organs. These abnormalities have been described as hypertrophy, hyperplasia or hamartomatous proliferations. In the male genital system, the excessive growth of smooth muscle in spermatic cord or paratesticular tissue is rare. In previously described cases, these lesions presented as masses but lacked the microscopic features of a neoplasm. We describe a complex multilocular cystic lesion composed of cystic transformation of the rete testis associated with smooth muscle proliferation mimicking intratesticular Leydig cell neoplasm. The lesion consists of three separate components: (1) cystic dilatation of the rete testis; (2) diffuse, interstitial smooth muscle proliferation with intraseptal expansion; and (3) extensive stroma with myxoid areas and scattered interstitial Leydig cells. These morphological findings, supported by a wide immunohistochemical panel, are consistent with cystic dilatation of the rete testis associated with smooth muscle hyperplasia, most probably of myoid origin. To the best of our knowledge, no similar complex lesion of the rete testis has yet been reported.     

The modulation of smooth muscle cell phenotype is an early event in human aorto-coronary saphenous vein grafts

Summary The morphological changes in human vein grafts occurring in the first days after a coronary bypass operation (CBP) are rarely reported in the literature. Sections of aorto-coronary vein grafts from 11 patients who died during the first 10 days after a CBP were obtained at autopsy. The number of vein grafts per patient ranged from 1 to 4, yielding a total of 28 vein grafts. The early changes in the vein grafts have been studied by light microscopy, immunohistochemistry, transmission and scanning electron microscopy. The study demonstrates that soon after grafting, the vein wall is infiltrated by polymorphonuclear leucocytes (PMN). At 24 h the endothelium shows extensive desquamation. The massive migration of PMN through the venous wall occurs simultaneously with the endothelial damage. The circular layer of the media is severely damaged, resulting in a loss of smooth muscle cells (SMC). The remaining SMC in this layer show a change toward the synthetic phenotype and a reduced expression of α-smooth muscle actin. These early changes in the SMC function may initiate the process of fibrosis in the intima and the media of the vein grafts.     

The costo-uterine muscle of the guinea-pig: A smooth muscle attaching the uterus to the last rib

Summary A conspicuous smooth muscle (of transverse sectional area comparable to that of the taenia coli) situated in the suspensorium ovarii and in the ligamentum ovarii proprium of the guinea-pig is described. The muscle is attached to the last rib, it reaches the medial side of the ovary, to which it is loosely attached; it passes beyond the caudal pole of the ovary (from which it receives additional muscle bundles), it approaches the oviduct, passing ventral to it, then it spreads around it and eventually reaches the distal end of the uterus; this muscle (which is here indicated as costo-uterine muscle) is in direct continuation with, or transforms itself into, the longitudinal outer coat of the uterus.     

Development of smooth muscle: ultrastructural study of the chick embryo gizzard

Summary The growth and differentiation of smooth muscle in the chicken gizzard were studied by electron microscopy from the 10th day in ovo to 6 months after hatching; during this period the organ grows 1000-fold in weight. At the earliest stage studied, smooth muscle cells, interstitial cells, and fibroblasts are immature but can already be clearly distinguished. The structural components of muscle cells develop in a characteristic sequence. Mitochondria are more abundant in immature muscle cells (8% in 14 days embryos and 7% in 19 days embryos) than in the adult (5%). Caveolae are virtually absent in the 11 day embryo; they become more common at the end of embryonic life, but continue to increase in relative frequency after hatching. Gap junctions appear around the 16th day in ovo as minute aggregates of connexons, which then grow in size, probably by addition of new connexons. In the earliest stages studied, myofilaments occupy 25% of the cell profile and are assembled into bundles accompanied by dense bodies and surrounded by loosely arranged intermediate filaments. By contrast, membrane-bound dense bands are scarce until the latter part of embryonic life, an observation suggesting that myofilament formation and alignment is not a process initiated near the cell membrane or directed by the cell membrane, and that only late in development bundles of myofilaments become extensively anchored to dense bands over the entire cell surface: at that time myofilaments occupy more than 75% of the cell volume. The muscle cells increase about four-fold in volume over the period studied; the 1000-fold increase in muscle volume is mainly accounted for by an increase in muscle cell number. Mitoses are found in the gizzard musculature at all embryonic ages with a peak at 17–19 days; they occur in muscle cells with a high degree of differentiation. These cells divide at a stage when they are packed with myofilaments and form junctions with neigh bouring cells: the mitotic process affects the middle portion of the cellm which takes up an ovoid shape and eventually divides, whereas the remaining portions of the cell do not differ in appearance from the surrounding muscle cells. At all stages of development the population of muscle cells has a uniform appearance (apart from the cells in mitosis), and the growth and differentiation seem to proceed at the same pace in all the cells. There are no undifferentiated cells left behind in the tissue for later development.     

Ultrastructure of airway smooth muscle

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.     

大鼠细小动脉平滑肌细胞分离培养的新方法

目的：探讨大鼠细小动脉平滑肌细胞分离培养的新方法。方法：取大鼠肺分支动脉,先切成小块进行胶原酶的消化,约 ８ ｈ,而后用含２０％小牛血清的 ＤＥＭＥ培养基贴块培养。结果：培养 ２４ ｈ,可见有大量细胞游出贴瓶底生长,７２ｈ已融合成片,呈典型的“谷峰”样长势。尚有少量内皮细胞,通过消化传代除去。取传第三代细胞进行抗α－ａｃｔｉｎ免疫组织化学染色,大量饮泡,基膜下有密斑,密体。免疫组化鉴定培养细胞纯度为９６％。结论：应用消化贴块法培养的大鼠平滑肌细胞,方法简单,结果可靠,具有应用价值。     

Myofibroblastoma of the breast with smooth muscle differentiation showing deletion of 13q14 region: Report of a case

Breast myofibroblastomas are rare benign mesenchymal tumors belonging to the group of stromal breast tumors composed of spindle-shaped cells and characterized by a broad morphologic spectrum. Among the different morphologic variants described, breast MFBs can show smooth muscle cell differentiation in very rare cases.     

Cytoskeletal mechanics in airway smooth muscle cells

Mechanical properties and contractility of airway smooth muscle tissue are largely responsible for airway narrowing and airway hyperresponsiveness in asthma. To explain these pathological phenomena, investigators have studied the mechanical behaviour of airway smooth muscle cells and its relationship to the underlying cellular biophysical and biochemical mechanisms. During the past decade, a growing body of evidence has indicated that a deformable intracellular polymer network, known as the cytoskeleton, plays a major role in transmitting and distributing mechanical forces within the cell and in their conversion into biochemical responses. We review here evidence suggesting that the tensed and crosslinked cytoskeletal lattice, the contractile apparatus, and the cytoskeleton–extracellular matrix interactions are key determinants of mechanical properties and mechanosensing of airway smooth muscle cells, with the mechanical distending stress of the cytoskeleton playing the central role.     

Endothelialization and patency of RGD-functionalized vascular grafts in a rabbit carotid artery model

To address the growing demand of small-diameter vascular grafts for cardiovascular disease, it is necessary to develop substitutes with bio-functionalities, such as anticoagulation, rapid endothelialization, and smooth muscle regeneration. In this study, the small-diameter tubular grafts (2.2 mm) were fabricated by electrospinning of biodegradable polymer polycaprolactone (PCL) followed by functional surface coating with an arginine-glycine-aspartic acid (RGD)-containing molecule. The healing characteristics of the grafts were evaluated by implanting them in rabbit carotid arteries for 2 and 4 weeks. Results showed that at both time points, all 10 of the RGD-modified PCL grafts (PCL-RGD) were patent, whereas 4 of the 10 non-modified PCL grafts were occluded due to thrombus formation. Scanning electron microscopy (SEM) data showed abundant platelets adhering on the surface of the midportion of the PCL grafts. In contrast, only few platelets were observed on the PCL-RGD surface, suggesting that RGD modification significantly improved the hemocompatibility of the PCL grafts. Histological analysis demonstrated enhanced cell infiltration and homogeneous distribution within the PCL-RGD grafts in comparison with the PCL grafts. Furthermore, immunofluorescence staining also showed a 3-fold increase of endothelial coverage of the PCL-RGD grafts than that of PCL grafts at those two time points. After 4-week implantation, 65.3 ± 7.6% of the surface area of the PCL-RGD grafts was covered by smooth muscle cell layer, which is almost 23% more than that on the PCL grafts. The present study indicates that RGD-modified PCL grafts exhibit an improved remodeling and integration capability in revascularization.     

Skinned smooth muscle: calcium-calmodulin activation independent of myosin phosphorylation

In chemically skinned chicken gizzard smooth muscle fibers investigated shortly after preparation, a contraction may be induced by calcium and calmodulin which is independent of myosin phosphorylation at intermediate Ca²⁺-concentrations. However, fibers stored for a prolonged period also contract in the absence of exogenous calmodulin and exhibit a close relationship between force development and myosin phosphorylation.     

Myosin composition and functional properties of smooth muscle from the uterus of pregnant and non-pregnant rats

The myosin heavy chain stoichiometry and the force-velocity relation have been determined in the myometrium of the non-pregnant and pregnant rat. The relative proportions of the slower migrating heavy chain (MHC₁) greatly exceeded that of the faster migrating heavy chain (MHC₂) as shown by electrophoresis on SDS 4%-polyacrylamide gels. The ratios of MHC₁/MHC₂ were 2.2/1 in the non-pregnant rats, 2.6/1 in the pregnant rat, and contrasted with 0.8/1 in the rat portal vein. This stoichiometry was unchanged by extracting the myosin from the smooth muscle as native myosin in a salt extract, as dissociated myosin using sodium dodecyl sulphate (SDS) or by isolating the native myosin first by a non-dissociating (pyrophosphate) electrophoresis step and subsequently analysing the protein bands on the SDS 4%-polyacrylamide gel. Although the unequal proportions of the heavy chains suggested the possibility that the native myosin molecule may be arranged as homodimeric heavy chains, no evidence for or against the existence of native myosin isoforms could be obtained by electrophoresing native myosin extracts on pyrophosphate-polyacrylamide gels. The force-velocity relations of the intact electrically stimulated myometrium from the non-pregnant and pregnant rats gave isometric force of 45 and 135 mN/mm² andV _max of 0.71 and 0.52 lengths/s (37°C) when measured at 95% of optimal length, whereas in chemically skinned uterine strips at 22°CV _max was 0.09 and 0.13 lengths/s, respectively. The length-force relationship was of similar shape in the non-gravid and gravid skinned tissues. The energetic tension cost (ATP-turnover/active stress) in skinned fibres was also similar. The mechanical and metabolic characteristics of the gravid and non-gravid uterus found in the present study do not suggest an obvious difference in the intrinsic properties of the myosin, although significant functional alterations in the tissue appear during pregnancy. This corresponds to the lack of a difference in the pattern of the heavy chains.     

A possible role of linked Na and Cl movement in active Cl uptake in smooth muscle

Smooth muscles actively accumulate Cl. Abolition of the transmembrane Na gradient has been shown to lead to a passive distribution of Cl. This suggested to us that Na and Cl may move together across the smooth muscle cell membrane, Cl accumulation resulting due to [Na]_i being less than [Na]_o. In favour of this view are the findings that Cl uptake is reduced in Na-free medium, Na uptake is reduced in Cl-free medium, reducing [Cl]_i reduces Na efflux, and increasing [Na]_i increases Cl efflux.     

Isolation of vascular smooth muscle cells from a single murine aorta

The vascular smooth muscle cell plays a significant role in many important cardiovascular disorders, and smooth muscle biology is therefore important to cardiovascular research. The mouse is critical to basic cardiovascular research, largely because techniques for genetic manipulation are more fully developed in the mouse than in any othermammalian species. We describe here a technique for isolating smooth muscle cells from a single mouse aorta. This technique is particularly useful when material is limiting, as is frequently the case when genetically modified animals are being characterized.     

A novel isoform of the smooth muscle cell differentiation marker smoothelin

Studies on smooth muscle cell differentiation and those on vascular development in mouse and humans have long been hampered by the lack of suitable markers. Here we describe a novel, large isoform of smoothelin, a structural protein of differentiated, contractile smooth muscle cells. The protein, which is highly conserved in mouse and humans, shows homology with other cytoskeleton-associated smooth muscle cell proteins and contains an actinin-type actin-binding domain. Northern blot analysis from various mouse organs identified short and long smoothelin mRNA forms, which exhibit distinct tissue expression patterns. The short form is highly expressed in visceral muscle tissues such as intestine and stomach and is not detectable in brain, while the long mRNA form is expressed in all vascularized organs. These results may provide new tools and approaches to study both smooth muscle cell differentiation and proliferative vascular disease. Received: 25 August 1998 / Accepted: 19 October 1998