Ontogeny of airway smooth muscle: structure, innervation, myogenesis and function in the fetal lung

Airway smooth muscle (ASM) is an integral component of the primordial lung. It differentiates from the mesenchyme as a ring of cells around the base of the epithelial bud that express smooth muscle-specific proteins. These rapidly form into interlocking bundles that progressively become wider and more compact along the bronchial tree to the trachea. Their orientation is perpendicular to the long axis of the airway. The ASM exhibits rhythmic contractility (i.e. it is a phasic-type smooth muscle) soon after formation, and the spontaneous airway narrowing shifts the lung liquid distally causing expansion of the tubule walls. This stretching is the mechanical stimulus to smooth muscle (SM) myogenesis and lung growth. Neural tissue, i.e. precursor ganglia interconnected by nerve trunks and smaller bundles, forms a sheath over the ASM layer with varicose fibres descending to the muscle. These are guided by glial-derived neurotrophic factor (GDNF) that appears to be produced by ASM. Maturation of neural tissue is slower than the ASM; functional cholinergic innervation is manifest by the early canalicular stage when most neurotransmitters appear.     

Development of the innervation and airway smooth muscle in human fetal lung

Human and porcine fetal airways have been shown to contract spontaneously from the first trimester, the latter also contracting in response to neural stimulation. Our object was to map immunohistochemically the innervation and its relationship to the airway smooth muscle (ASM) in the human fetal lung from early gestation to the postnatal period. Whole mounts of the bronchial tree were stained with antibodies to the pan-neuronal marker protein gene product 9.5, the Schwann cell marker S-100, and the ASM contractile protein alpha-actin, and imaged using confocal microscopy. By the end of the embryonic period (53 d gestation), the branching epithelial tubules in the primordial lung were covered with ASM to the base of the terminal sacs. An extensive plexus of nerve trunks containing nerve bundles, forming ganglia, and Schwann cells ensheathed the ASM. By 16 wk (canalicular stage), maturation of the innervation was advanced with two major nerve trunks running the length of the bronchial tree, giving rise to varicosed fibers lying on the ASM. An extensive nerve plexus in the mucosa was also present. The distal airways of infants who had died of Sudden Infant Death Syndrome were also covered with smooth muscle and were well innervated. Thus, an essentially complete coat of ASM and an abundant neural plexus ensheathing the airways are an integral part of the branching epithelial tubules from early in lung development.     

Fine structure of cystosarcoma phyllodes with reference to smooth muscle tumors

The fine structure of the stomal component of cystosarcoma phyllodes was compared with that of intracanalicular fibroademona, leiomyoma and leiomyosarcoma. The stromal cells of fibroadenoma was intermediate between fibroblasts and smooth muscle cells and may be designated as myofibroblasts. The stromal cells of cystosarcoma phyllodes have fulfilled the ultrastructural criteria for smooth muscle cells. Therefore, cystosarcoma phyllodes should be regarded as smooth muscle tumors or those of myoepithelial origin.     

Vascular leiomyosarcoma: Clinicopathology and immunohistochemistry with special reference to a unique smooth muscle phenotype

Vascular leiomyosarcoma (LMS) is the least frequent type of soft‐tissue LMS and has not been fully evaluated immunohistochemically. The aim of the present study was therefore to examine four cases of vascular LMS, paying special attention to the immunohistochemical expression of smooth muscle markers. All the patients were female; two tumors were located within the inferior vena cava, one within the saphenous vein, and one within the external iliac vein. All the tumors displayed well–moderately differentiated smooth muscle morphology. On immunohistochemistry all four vascular LMS were diffusely and strongly reactive to actins, calponin, and h‐caldesmon, whereas desmin was diffusely stained in one tumor, only focally positive in two, and negative in one. The walls of the blood vessels contiguous to the tumors contained desmin‐ and h‐caldesmon‐positive smooth muscle cells in two cases. In contrast, among 43 non‐vascular LMS (20 well–moderately differentiated and 23 poorly differentiated or other types), 38 were variably positive for desmin, whereas 21 were negative for h‐caldesmon. These findings suggest that the vascular LMS displays a distinctive phenotype of smooth muscle differentiation according to histopathogenesis.     

Mechanical components of rat intestinal villi as revealed by ultrastructural analysis with special reference to the axial smooth muscle cells in the villi

The ultrastructure of the rat intestinal interstitium with regard to the mechanical components was analyzed from a functional viewpoint utilizing serial horizontal as well as longitudinal sections through the lamina propria mucosae, including both villi and crypts. The axial smooth muscle cells in the villi (villus-axial SMs) exhibited different configurations at various levels of the wall. They were separated from the voluminous fluid-filled spaces by sheet-like processes of fibroblasts in the upper part of the intravillous interstitium, formed a sheet around the central lymphatics, and were covered by the sheet-like processes of fibroblasts in the lower part of the intravillous interstitium. These villus-axial SMs were poorly developed and associated with the lymphatic walls in the upper part of the pericryptal interstitium; they were tapered and connected to microtendons composed of fascicles of longitudinal collagen fibrils in the lower part of pericryptal interstitium. At the apical termination, the villus-axial SMs were connected to myofibloblasts, which sent off many processes into the subepithelial meshwork layer of fine cell processes and extracellular matrices. The villus-axial SMs possibly develop longitudinal tension against the intravillous hydraulic pressure developing from the transepithelial absorption through the intestinal epithelium.     

Developmental changes in intracellular pH buffering power in smooth muscle

 Intracellular pH (pH_i) is known to modulate contraction. Neonatal tissues can differ from adult tissue in contractile response to stimuli known to alter pH_i e.g. hypoxia. Changes of pH are attenuated by buffering, thus any difference in buffering power (β) between tissues could affect their functional response to pH_i perturbation. Similarly the extent to which any extracellular pH (pH_o) alteration is transmitted into a pH_i change will also influence function. We have therefore determined the intrinsic β and effect of pH_o change on pH_i in neonatal and adult ureteric, uterine and gastric smooth muscles using the pH-sensitive fluorophore carboxy-SNARF. β was found to be similar in the three adult tissues, but there were significant differences between neonatal tissues. In contrast, we found little difference in the amount of pH_i change produced by pH_o change between neonatal and adult tissues from the same smooth muscle, but a difference between smooth muscles. These data highlight significant differences between smooth muscles and their developmental state, which may contribute to different degrees of protection when pH is perturbed. Received: 17 October 1997 / Received after revision: 27 November 1997 / Accepted: 28 November 1997     

Morphological and functional features of endodermal cells in rat yolk sac, with special reference to the fetal macrophage differentiation

Morphological and functional features of the yolk sac endodermal cells with special reference to the fetal macrophage differentiation were investigated morphologically under the light and electron microscopes and immunologically with the antigen phenotypic analysis and the phagocytic activity-test, using the syngeneic DA rat-embryos from 8 to 16 days of gestation. Based on the staining property with toluidin blue and the ultrastructural features, the endodermal layer from day 8 to 16-yolk sacs has been known to consist of two kinds of cell type; 10% "clear" cells with clear cytoplasm and 90% "dark" cells with dark cytoplasm. Numerous primary lysosomes, phagolysosomes, lipid droplets and coated vesicles distributed preferentially in the supranuclear portion of endodermal cells. A broad intercellular space was found between "clear" cells and "dark" cells, indicating the loose intercellular binding. It was often found that "clear" cells tend to migrate from the endodermal layer into the mesenchymal layer, where the poor development of basement membrane was seen between them. Cells phagocytosing red blood cells, that resemble morphologically "clear" cells, were also observed in the fetal liver. At ten hours after latex-injection into the yolk sac cavity of 14 days embryos, some cells which phagocytosed latex beads in their cytoplasm were found in the endodermal layer, and also in the liver tissue and loose connective tissue of fetus. These cells were stained positively with monoclonal antibody Mar3 which recognizes preferentially rat-mononuclear phagocyte system. In vitro-latex uptake of separated endodermal cells was also demonstrated by the culture-study of endodermal cell suspension. The present findings indicate that the yolk sac-endodermal layer derived from the proximal endoderm consists of at least two kinds of cell-population with a great similarity to tissue macrophages in morphological and functional senses, and support the concept that some cell-populations of endodermal layer may migrate into fetal tissue and are closely related to the differentiation of fetal macrophages and their precursors.     

Hyaluronan in the rat with special reference to the skin

The total hyaluronan content has been determined in rats. The animals were frozen and sectioned in a cryostat before digestion with papain and pronase. The hyaluronan content was determined by a specific radioassay and it was found that 250 g rats contained 40-60 mg of the polysaccharide. The recovery from the preparation procedure was close to 100%, as determined from tracer experiments. More than half of the hyaluronan was found in skin, approximately one quarter in the skeleton and supporting structures and less than one tenth in skeletal muscle. Based on calculated lymph flow and lymph concentration of hyaluronan, it seems that a significant fraction of the total hyaluronan in skin (greater than 1%) is removed via the lymphatics in a 24 h period. An attempt was made to isolate undegraded hyaluronan from rat skin by gentle methods giving full recovery in order to estimate the molecular weight of the polysaccharide. Hyaluronan was recovered quantitatively, but as determined from added tracer, it had been degraded. Correction for the estimated degradation gave a molecular weight of several millions for the endogenous hyaluronan.     

Obesity, smooth muscle, and airway hyperresponsiveness

Both asthma and obesity are large and growing public health issues. Mounting evidence now implicates obesity as a major risk factor for asthma, thus linking these 2 major epidemics. Moreover, both in human subjects and in mice, obesity appears to predispose toward airway hyperresponsiveness. This review describes potential mechanisms whereby obesity might modify airway smooth muscle function to explain these observations. These mechanisms include both static and dynamic mechanical factors attributable to decreases in functional residual capacity and decreases in tidal volume that are observed in the obese. They include also obesity-related changes in lung development, chronic systemic inflammation (including increased serum levels of inflammatory cytokines and chemokines), and adipocyte-derived factors, including leptin, adiponectin, and plasminogen activator inhibitor.     

Isolation of silica-dependent protein from rat lung with special reference to development of fibrosis

Silicosis was produced experimentally in rats by single intratracheal injections of various doses of SiO2 dust. The weight of the lungs as well as the contents of total nitrogen, collagen, nucleic acids (especially RNA), and lipids increased in accordance with the dose and the time interval. Fibrogenic stimulation in vitro was shown by the supernatant of the homogenized lung in the incorporation of proline into incubated granulation tissue or lung fibroblasts. The fibrogenic factor-activity depended more on the time interval after the injection than on the SiO2 dose. Electrophoresis of the soluble proteins in the silicotic rat lungs showed a protein of 16,000 Da, which was dependent on the time interval following SiO2 administration as well as on the dose itself, and which originated from macrophages. This protein was purified by repeated gel-filtration chromatography. It stimulated collagen synthesis in granulation-tissue cells at a concentration of about 10(-10) M in a dose-dependent way. It was acidic by amino acid composition but differed from calmodulin which also increased collagen synthesis in granulation-tissue cells in vitro. The ability of non-fractionated macrophage preparations to stimulate the incorporation of proline into collagen correlated inversely with the gross alkaline RNase activity.     

Development of neural tissue and airway smooth muscle in fetal mouse lung explants: a role for glial-derived neurotrophic factor in lung innervation

We have characterized the distribution of neural tissue and its primary target tissue, airway smooth muscle (ASM), in an in vitro mouse model of early lung development comprising left lung lobes at embryonic Day 12, cultured for 2 or 5 d. Neural tissue was detected with antibodies to protein gene product 9.5 (PGP 9.5), synapsin, and p75NTR (the low-affinity neurotrophin receptor), and smooth muscle with an antibody to alpha-actin. Imaging by confocal microscopy revealed few PGP 9.5-positive neurons at the start of culture; after 2 d clusters of neurons and nerve fibers had appeared along the lobar bronchus and after 5 d along the secondary and tertiary branches. Neural tissue did not just follow the smooth muscle-covered tubules, as seen in vivo, but also grew outside the lobes onto a wide layer of alpha-actin-positive cells, suggesting that smooth muscle may express a trophic factor that attracts nerves. Explants cultured with glial-derived neurotrophic factor (GDNF) exhibited a striking increase in the amount of p75NTR- and PGP 9.5-positive tissue outside the lobes, whereas GDNF-impregnated beads attracted neuronal precursors and influenced the direction of neurite extension. We show that the mouse lung explant is suitable for investigating trophic signals involved in pulmonary innervation and that GDNF may have a role in the early innervation of the developing airways.     

Electron microscopic observations on the morphogenesis of the albino rat lung,with special reference to pulmonary epithelial cells

Electron microscopic observations of developing albino rat lung provide further evidence for the endodermal origin of the type I and II pulmonary epithelial cells, and for the mesodermal origin of the interstitial pulmonary cells. Cytoplasmic glycogen increased in the fetal endodermal cells from day 16 to the twentieth day of gestation. Further development revealed a decrease of this substance in the differentiating pulmonary epithelial cells, and a concurrent increase in the mesodermal interstitial pulmonary cells, these being cells formerly characterized by their high lipid content. A continuous basement membrane delineated the mesodermal and endodermal components of the developing rat lung from day 16 to the third postnatal day. Tight junctions between adjacent endodermal epithelial cells were present throughout this same gestational period. Lamellar bodies, which are characteristic of the type II pulmonary epithelial cell, may be found simultaneously with large quantities of cytoplasmic glycogen. This feature is considered characteristic of fetal endodermal components. A definite continuum of ultrastructural changes may be traced from the endodermal, columnar epithelial cells to the definitive type I and II pulmonary epithelial cells. Comparable observations for the mesodermal components reveal a progression leading to mature interstitial pulmonary cells.     

Expression of thrombomodulin in human aortic smooth muscle cells with special reference to atherosclerotic lesion types and age differences

Expression of thrombomodulin (TM) in atherosclerotic lesions of the human aorta (8 cases of diffuse intimal thickening, 4 fatty streaks, 11 atheromatous plaques, and 5 fibrous plaques) as well as in undiseased aortas of 5 infants obtained at autopsy was studied immunohistochemically using a novel polyclonal antibody against human TM. TM was expressed in intimal smooth muscle cells (SMC) besides endothelial cells and foamy macrophages in almost all patients (26/28). In addition, medial SMC in adult cases over 27 years of age expressed TM. In young adults with diffuse intimal thickening under 26 years of age, medial SMC showed no TM expression whereas intimal SMC did show it. Both intimal and medial SMC in infants showed no TM expression. An immunofluorescence method showed TM expression in cultured adult human SMC. These findings indicate that TM expression in SMC may depend on patient age as well as lesion type of atherosclerosis.     

血小板活化因子对大鼠气道平滑肌细胞的促增殖作用

目的：研究血小板活化因子(platelet-activating factor, PAF)对离体培养的大鼠气道平滑肌细胞(airway smooth muscle cell, ASMC)增殖的影响。方法： 离体培养大鼠气道平滑肌细胞，细胞分为对照组、PAF组；后者又 分为10-6、10-7、10-8、10-9 mol·L-1 4小组，MTT(四唑盐)比色法检测细胞增殖活力并确定PAF最佳作用浓度；用最佳作用浓度刺激ASMC 12 h、24 h、36 h以流式细胞仪测定细胞周期，免疫细胞化学染色(SABC 法)检测该PAF刺激48 h后细胞核增殖抗原(PCNA)的表达。 结果： PAF在10-6-10-9 mol·L-1范围内均能促进ASMC的增殖(P<0.01)，且10-7 mol·L-1时增殖作用最强；用这一最佳浓度的PAF干预12 h后，G0/1期细胞百分比(68.67%)明显低于对照组(85.57%)(P<0.01)；PCNA免疫细胞化学染色发现10-7mol·L-1的PAF作用48 h后，ASMC的PCNA表达率为(71.05±1.22)%，显著高于对照组(53.27±2.56)% (P<0.05)。结论： PAF能以时间依赖方式促进ASMC的增殖，但该作用并不表现为浓度依赖性。     

血小板活化因子促进大鼠气道平滑肌细胞增殖及其作用机制

目的研究血小板活化因子（PAF）对大鼠气道平滑肌细胞（ASMC）增殖活性的影响，并探讨其作用机制。方法以10^-7mol／LPAF作为刺激因素，以20mmol／LN-乙酰半胱氨酸（NAC）进行干预。用四唑盐（MTT）比色法检测细胞增殖活力；流式细胞仪测定细胞周期；免疫细胞化学染色（SABC法）检测细胞核增殖抗原（PCNA）的表达；EMSA法检测ASMC中NF-κB的活性。结果发现PAF能显著促进ASMC的增殖，明显增加细胞增殖指数及其PCNA阳性表达率，并使ASMC细胞核内NF-κB活性明显增加。预先用NAC干预可显著缓解上述变化（P〈0．05）。结论PAF能促进ASMC的增殖，这一调控部分是通过激活NF-κB通路而发挥作用。提示PAF是气道重建的重要刺激因子。     

Isolation of silica-dependent protein from rat lung with special reference to development of fibrosis.

Silicosis was produced experimentally in rats by single intratracheal injections of various doses of SiO2 dust. The weight of the lungs as well as the contents of total nitrogen, collagen, nucleic acids (especially RNA), and lipids increased in accordance with the dose and the time interval. Fibrogenic stimulation in vitro was shown by the supernatant of the homogenized lung in the incorporation of proline into incubated granulation tissue or lung fibroblasts. The fibrogenic factor-activity depended more on the time interval after the injection than on the SiO2 dose. Electrophoresis of the soluble proteins in the silicotic rat lungs showed a protein of 16,000 Da, which was dependent on the time interval following SiO2 administration as well as on the dose itself, and which originated from macrophages. This protein was purified by repeated gel-filtration chromatography. It stimulated collagen synthesis in granulation-tissue cells at a concentration of about 10(-10) M in a dose-dependent way. It was acidic by amino acid composition but differed from calmodulin which also increased collagen synthesis in granulation-tissue cells in vitro. The ability of non-fractionated macrophage preparations to stimulate the incorporation of proline into collagen correlated inversely with the gross alkaline RNase activity.