Altered cytoskeleton in smooth muscle of aganglionic bowel.
Smooth muscle cells possess the contractile apparatus of actin and myosin filaments and the cytoskeleton with the membrane skeleton, which provides the interface between the contractile machinery on the inside of the cell and the extracellular matrix on the outside, to which force must be transmitted. The cytoskeletal system serves as a structural framework that surrounds and supports the contractile apparatus in the body of the cell and has 3 major domains, which have several protein components. One domain consists of the contacts between the sarcolemma and the junctional sarcoplasmic reticulum (cytoskeletal domain), another contains caveolae (caveolae are microdomains of the plasma membrane that have been implicated in signal transduction), and the third contains adherens junctions, which are also referred to as membrane-associated dense bodies. (8-11) Dystrophin is a subsarcolemmal protein of the caveolar domain with a double adhesion property, one between the membrane elements and the contractile filaments and the other between the cytoskeletal proteins and the extracellular matrix via the laminin-binding system. (12) It has been suggested that dystrophin plays a role in smooth muscle cells similar to that of the synapses in neuromuscular junctions. (13) Desmin, the intermediate filament protein of skeletal muscle fibers, cardiac myocytes, and certain smooth muscle cells, is a part of the cytoskeletal domain linking Z-bands with the plasmalemma and the nucleus. (14,15) Vinculin is a functionally related protein of adherent junctions, structures by which cells make contacts with neighboring cells or to the extracellular matrix. At these sites, the actin cytoskeleton of smooth muscle cells is anchored to the plasma membrane. (8,9,11,16) Although the abnormalities of enteric nerves, including adrenergic, cholinergic, nonadrenergic noncholinergic (peptidergic), and nitrergic nerves, have been suggested to be the cause of bowel dysfunction in this condition, the exact pathogenesis of HD is not fully understood. (1-3) To our knowledge, the literature contains no information regarding the cytoskeleton of the smooth muscle cells in aganglionic bowel. The present study was undertaken to examine the distribution of cytoskeleton in the smooth muscle of aganglionic HD bowel.
MATERIALS AND METHODS
Full-thickness bowel specimens were obtained from 8 patients with rectosigmoid HD who underwent pull-through operations. The diagnosis of HD was confirmed preoperatively from suction biopsies and postoperatively from the resected bowel samples by acetylcholinesterase histochemistry and PGP 9.5 immunohistochemistry. Normal colon specimens were collected at the time of bladder augmentation from 4 patients, which acted as controls.
Fresh bowel specimens were rinsed, 1 X 1-cm pieces were cut and mounted unfixed into freezing medium (Leica, Nussloch, Germany), and the specimens were then frozen in liquid nitrogen. Specimens were embedded in paraffin after 8 hours' fixation in Zamboni solution.
Five-micrometer serial sections were cut from the paraffin and the fresh frozen blocks and were placed on gelatin-coated glass slides. The fresh frozen sections were incubated at room temperature for 20 minutes in 10% bovine serum albumin to prevent nonspecific binding. Paraffin sections were deparaffinized and blocked with 10% bovine serum albumin.
The primary antibodies were monoclonal anti-desmin (diluted 1:20; Chemicon, Temecula, Calif), monoclonal anti-dystrophin (1: 50; Chemicon), and monoclonal anti-vinculin (1:40; Chemicon).
The secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunoglobulin (1:20; Dako, Glostrup, Denmark), FITC-conjugated rabbit anti-mouse immunoglobulin (1:20; Dako), Texas-Red-conjugated goat anti-mouse and goat anti-rabbit immunoglobulins (1:50; Molecular Probes, Leiden, Netherlands). No immunostaining was observed when primary antisera were omitted or replaced with normal serum in controls.
Incubation with the primary antibody took place overnight at 4[degrees]C. After being rinsed in phosphate-buffered saline, the slides were incubated with secondary antibody for 60 minutes at room temperature. Finally, the sections were mounted in fluorescein mounting medium (Dako) and covered with glass. The slides were kept at 4[degrees]C to minimize loss of fluorescein activity until analysis with confocal laser scanning microscopy.
The sections were observed using an upright laser scanning confocal microscope (BIO-RAD 2000, Hamil Hamsted, United Kingdom) with immersion objectives (X40 numerical aperture 0.45, NPL Fluotar, or X60 numerical aperture 0.75). Tissue sections were excited using krypton/argon laser with excitation and barrier filters set for individual fluorophores according to their specific excitation-emission spectra ([lambda] = 568 nm, 488 nm, 647 nm). The emitted light was detected by a photomultiplier tube and converted via an analogue-to-digital converter (BIO-RAD, MRC 1024) into a digital pixelated image (512 X 512 picture elements). The detection pinhole was set for use with X40 and X63 objectives accordingly. Offset and gain settings were determined at the start of each experiment and kept constant throughout, with laser power recorded each time.
Quantification of Results
The density of immunostaining was graded as follows: -, non-immunostaining; [+ or -], occasional immunostaining; +, moderate immunostaining; and ++, positive immunostaining. Two independent investigators evaluated the immunostaining semiquantitatively. When the grades were not identical, sections were reviewed and a consensus was reached.
Normal Bowel and Ganglionic Segment of HD Bowel
All cytoskeletal proteins displayed strong immunoreactivity (Table). Dystrophin colocalized in its typical appearance at the periphery of the normal smooth muscle fibers (Figure 1, A). Desmin immunoreactivity was evenly distributed throughout the cell, with strong or moderate staining (Figure 2, A). Strong vinculin immunoreactivity was observed in the circular and longitudinal smooth muscle layer of the normal and ganglionic bowel specimens (Figure 3, A).
[FIGURES 1-3 OMITTED]
Aganglionic Segment of HD Bowel
Dystrophin immunoreactivity was weak or totally absent in the aganglionic bowel (Figure 1, B; Table). Desmin immunoreactivity was weak or totally absent in the smooth muscle cells (Figure 2, B). Vinculin expression was weak in some areas, but was absent in smooth muscle cells of both the circular and longitudinal muscle layer of the aganglionic segment of HD bowel (Figure 3, B).
The muscle cell cytoskeleton consists of proteins and structures whose primary function is to link, anchor, or tether structural components inside the cell. (8,9) Unfortunately, this role has sometimes evoked the concept that the cytoskeleton of muscle cells is relatively static and less interesting than the contractile machinery. (4-6) However, 2 important attributes of the cytoskeleton are strength of various attachments and flexibility to accommodate the changes in cell geometry that occur during contraction. Well-documented observations that smooth muscle cells can contract to 60% of rest length, and can return to their rest length with the help of their internal structure, suggest that the cytoskeleton must be highly adaptable to cell-shape changes. (17)
Smooth muscle cells possess a contractile apparatus consisting of actin and myosin filaments and the cytoskeleton. The cytoskeleton with the membrane skeleton surrounds and supports the contractile apparatus and forms an association with the plasmalemma. (18) A major component of the cytoskeleton is the system of intermediate filaments that is an insoluble network and that serves a structural role in the cell body. The proteins that polymerize into cytoplasmic intermediate filaments belong to a heterogeneous family. In visceral smooth muscles, the intermediate filaments are composed solely of desmin, which belongs to the vimentin-like family and is one of the earliest known myogenic cytoskeletal markers. Desmin intermediate filaments surround the Z-disks, interlink them together, and integrate the contractile apparatus with the sarcolemma and the nucleus. According to this finding, desmin may mediate signal transduction and transport processes from the cell surface to the nucleus. (15,19,20)
To investigate the function of desmin in all muscle types in vivo, desmin-null mice were generated through homologous recombination. (21) Desmin-null mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle cells, beginning early in their postnatal life. Histologic and electron microscopic analysis of heart, skeletal, and smooth muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondrial number and organization, and loss of nuclear shape and positioning. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium, accompanied by extensive calcification. There is a direct correlation between severity of damage and muscle use, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. Studies so far have demonstrated that desmin is absolutely necessary for muscle differentiation in vitro and plays an essential role in the maintenance of myofibril, myofiber, and whole muscle structural and functional integrity. (15,20,21)
Dystrophin is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily and colocalizes in the membrane skeleton's caveolar domain. Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Dystrophin binds to subplasmalemmal actin filaments via its amino-terminal domain. The carboxy terminus of dystrophin binds to a plasma membrane anchor, [beta]-dystroglycan, which is associated on the external side with the extracellular matrix receptor, [alpha]-dystroglycan, which binds to the basal lamina proteins laminin 1, laminin 2, and agrin. In the muscle, the dystroglycan complex is associated with the sarcoglycan complex, which consists of several glycosylated integral membrane proteins. (8,9,11-13) The absence or functional deficiency of the dystrophin cytoskeleton is the cause of several types of muscular dystrophies, including the lethal Duchenne muscular dystrophy, one of the most severe and most common genetic disorders. (22) It is hypothesized that its absence in Duchenne muscular dystrophy may result in instability of the muscle cell membrane with resultant ingress of calcium, an increase in intracellular calcium, and cell death. The dystrophin complex is believed to stabilize the plasma membrane during cycles of contraction and relaxation. Dystrophin and several types of dystrophin variants are also present in extramuscular tissues, for example, in distinct regions of the central nervous system, including the retina. Absence of dystrophin from these sites is believed to be responsible for some extramuscular symptoms of Duchenne muscular dystrophy, for example, mental retardation and disturbances in retinal electrophysiology. (23)
Protein compositions of the dense plaques of the membrane skeleton belong to the family of adherens-type junctions, and vinculin is one of the functionally related protein structures in which cells make contacts to neighboring cells or to the extracellular matrix. (6,9,11,18) At these sites, the non-muscle actin cytoskeleton of animal cells is anchored to the plasma membrane. Junction assembly and disassembly are coordinated in processes as different as mitosis, cell movement, and tissue formation. Since adherens junctions are assembled from a large number of proteins, these molecules have to be coordinately activated and spatially regulated. Vinculin is also thought to serve a role as a tumor suppressor, suggesting that it has a regulatory function in addition to its structural role. (9,18)
Our study demonstrates that smooth muscle cells in both circular and longitudinal muscle layers from normal colon and from the ganglionic segment of HD patients have a normal cytoskeleton. The lack of proteins from the 3 major domains of the cytoskeleton and membrane skeleton in the aganglionic bowel suggests an altered cytoskeleton in the contracted bowel segment. The absence of dystrophin in the aganglionic bowel suggests that the caveolar domain is not completely developed in these cells and may be responsible for the interruption of linkage with the cytoplasmic contractile elements and the alteration in cell matrix communications. The lack of vinculin, a member of the adherent junction proteins, would suggest that signal transduction between the cell and extracellular matrix is not intact in the affected bowel in HD. The lack of desmin in the aganglionic colon results in a lack of muscle integrity and weakens the mechanochemical signaling within the muscle cell.
In conclusion, our results demonstrate that in patients with HD, cell cytoskeleton-extracellular matrix relationships are altered because of the absence of the characteristic proteins of the 3 well-determined cytoskeletal domains. An important unanswered question remains: what is the relationship between the hypertrophic nerve trunks and the structurally and functionally abnormal smooth muscle cells in the aganglionic bowel?
Immunostaining of Cytoskeletal Proteins in Normal, Ganglionic, and Aganglionic Bowel From a Patient With Hirschprung Disease (HD) Normal HD Ganglionic ++ + [+ or -] - ++ + [+ or -] - Dystrophin 4 4 ... ... 3 4 1 ... Vinculin 6 2 ... ... 4 4 ... ... Desmin 4 4 ... ... 4 4 ... ... HD Aganglionic ++ + [+ or -] - Dystrophin ... ... 6 2 Vinculin ... ... 1 7 Desmin ... ... 3 5 * ++ indicates strong; +, moderate; [+ or -], weak; and -, nil.
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Accepted for publication January 2, 2002.
From the Children's Research Centre, Our Lady's Hospital for Sick Children, Dublin, Ireland (Drs Nemeth, Rolle, and Puri); and the Department of Paediatric Surgery, University of Szeged, Szeged, Hungary (Dr Nemeth).
Reprints: Prem Puri, MS, FRCS, FRCS(Ed), Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland (e-mail: firstname.lastname@example.org).
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|Author:||Nemeth, Laszlo; Rolle, Udo; Puri, Prem|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Jun 1, 2002|
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