A neo-esophagus reconstructed by cultured human esophageal epithelial cells, smooth muscle cells, fibroblasts, and collagen

The scientists involved in this study attempted to develop an artificial esophagus constructed of autologous cells grown by cell culture methods on an extracellular matrix. An artificial esophagus consisting of human esophageal epithelial cells, dermal fibroblasts, and smooth muscle cells isolated from the aortic media, was attempted. The purpose of this study was to examine whether smooth muscle cells could be used in the transforming matrix. Human fibroblasts were embedded in Type I collagen superimposed on the collagen layer of smooth muscle cells. Next, human esophageal epithelial cells were cultured on the collagen layer of the fibroblasts. The resulting collagen sheets were cultured in vitro for 1 week, then transplanted on the latissimus dorsi muscles of athymic rats. The sheets were examined histologically at 1 and 2 weeks using hematoxylin eosin and immunologic stain methods (antiactin antibody). At the end of 2 weeks after transplantation, on microscopic observation of the collagen sheets, it appeared that the epithelial layer, the submucosal tissue layer, and the proper muscle layer had been reconstructed. Additionally, the authors successfully isolated smooth muscle cells from the media of the left gastric artery as a surgical specimen by explant cell culture. The ability to transform collagen sheets consisting of esophageal epithelial cells, fibroblasts, and smooth muscle cells from a surgical specimen into a luminal structure may enable clinical application of the artificial esophagus.     

A light microscope study of the distribution of muscle in the frog esophagus and stomach.

The present study reports light microscopical observations of the distribution of muscle in the esophagus and stomach of both the bull frog (Rana catesbeiana) and the African clawed frog (Xenopus laevis). The external muscle coat of the upper half of the esophagus in both species had several collagen coated bundles of striated muscle fibres around the circumference. These striated muscle bundles ran longitudinally from the pharynx to around the vicinity of the center of the esophagus. Beneath these striated muscle bundles was an inner circular layer of smooth muscle. In both species, the inner circular layer of smooth muscle was particularly thick in the region close to the pharynx. In the bull frog, the lower half of the esophagus lacked striated muscle. However, the circular smooth muscle layer, extending from the upper half of the esophagus, was also observed throughout the lower half of the esophagus. An outer longitudinal layer of smooth muscle developed towards the terminal portion of the esophagus such that in this region, both outer longitudinal and inner circular layers of smooth muscle were observed. Similarly in the African clawed frog, the inner circular layer of smooth muscle was continuous along the full length of the esophagus. Again, no striated muscle bundles were observed in the lower half of the esophagus. However, the outer longitudinal layer of smooth muscle was seen to develop in the middle region of the esophagus. Its muscle layer extended to the terminal portion of the esophagus. Thus, both outer longitudinal and inner circular layers of smooth muscle were observed throughout the lower half of the esophagus. In both frogs, the thickness of the outer longitudinal and inner circular layers of smooth muscle changed before and after the esophago-gastric junction. In both frogs, no muscularis mucosa was observed in the esophageal wall. However, in the lower half of the esophagus of the African clawed frog, small bundles of smooth muscle were observed here and there in the submucosa. A fully developed muscularis mucosa with both outer longitudinal and inner circular layers was observed in the upper stomach of both frogs.     

The expression of nestin delineates skeletal muscle differentiation in the developing rat esophagus

The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.     

Smooth muscle persists in the muscularis externa of developing and adult mouse esophagus

Following initial patterning as differentiated smooth muscle (SM) cells, the muscularis externa of the murine esophagus is replaced by skeletal muscle, but the mechanism underlying this process is controversial. The hypothesis that committed SM cells transdifferentiate into striated muscle is not consistent with fate mapping studies. Similarly, apoptosis does not fully explain the process. Using immunohistochemical techniques and transgenic mice that express eGFP and Cre-recombinase exclusively in SM, we have identified a population of remnant SM cells that persist throughout the developing and mature murine esophagus. These cells display an atypical phenotype, are not associated with microvasculature, but are often apposed to cKit positive, interstitial cells of Cajal. The absolute length of the SM component of the developing esophagus remains constant during a period when total esophageal length increases 4-fold, resulting in a small maintained distal segment of smooth muscle. Esophageal SM cells fail to express myogenin during development, and striated muscle cell precursors expressing myogenin fail to express specific SM cell markers, indicating that they did not transdifferentiate from SM cells. Moreover, smooth muscle-specific myogenin inactivation has no effect on esophageal skeletal myogenesis. Taken together, our results provide an alternative hypothesis regarding the fate of SM cells in the developing murine esophagus, which does not invoke apoptosis or transdifferentiation.     

Ultrastructural analysis of the smooth-to-striated transition zone in the developing mouse esophagus: emphasis on apoptosis of smooth and origin and differentiation of striated muscle cells.

The exact mechanism of smooth-to-striated muscle conversion in the mouse esophagus is controversial. Smooth-to-striated muscle cell transdifferentiation vs. distinct differentiation pathways for both muscle types were proposed. Main arguments for transdifferentiation were the failure to detect apoptotic smooth and the unknown origin of striated muscle cells during esophageal myogenesis. To reinvestigate this issue, we analyzed esophagi of 4-day-old mice by electron microscopy and a fine-grained sampling strategy considering that, in perinatal esophagus, the replacement of smooth by striated muscle progresses craniocaudally, while striated myogenesis advances caudocranially. We found numerous (1) apoptotic smooth muscle cells located mainly in a transition zone, where smooth intermingled with developing striated muscle cells, and (2) mesenchymal cells in the smooth muscle portion below the transition zone, which appeared to give rise to striated muscle fibers. Taken together, these results provide further evidence for distinct differentiation pathways of both muscle types during esophagus development.     

Structure of the esophagus in the adult opossum, Didelphis virginiana.

The structure of the esophagus has been studied in the adult opossum, Didelphis virginiana. A thickening of both layers of the muscularis externa occurs at the origin of the esophagus and may represent the upper esophageal sphincter; a massive expansion of the muscularis mucosae occurs in the region of the lower esophageal sphincter. The distribution of striated, mixed and smooth muscle in the muscularis externa differs in the inner and outer layers and elements of the myenteric plexus are found to occur even in the region of striated muscle; however, the ganglia of this plexus become much more prominent as smooth muscle makes its appearance. Esophageal glands are found in the lamina propria where they are confined to the 2 ends. They are especially prominent at the distal end where they are responsible for the formation of permanent transverse folds. Similar glands are found in the submucosa, scattered throughout the length of the esophagus but distally, in the region of the transverse folds, the submucous glands disappear. In both of these layers, the glands contain mucous, serous and myoepithelial cells.     

The neural regulation of the mammalian esophageal motility and its implication for esophageal diseases

In contrast to the tunica muscularis of the stomach, small intestine and large intestine, the external muscle layer of the mammalian esophagus contains not only smooth muscle but also striated muscle fibers. Although the swallowing pattern generator initiates the peristaltic movement via vagal preganglionic neurons that project to the myenteric ganglia in the smooth muscle esophagus, the progressing front of contraction is organized by a local reflex circuit composed by intrinsic neurons similarly to other gastrointestinal tracts. On the other hand, the peristalsis of the striated muscle esophagus is both initiated and organized by the swallowing pattern generator via vagal motor neurons that directly innervate the muscle fibers. The presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the esophagus had been enigmatic and neglected in terms of peristaltic control for a long time. Recently, the regulatory roles of intrinsic neurons in the esophageal striated muscle have been clarified. It was reported that esophageal striated muscle receives dual innervation from both vagal motor fibers originating in the brainstem and varicose intrinsic nerve fibers originating in the myenteric plexus, which is called ‘enteric co-innervation’ of esophageal motor endplates. Moreover, a putative local neural reflex pathway that can control the motility of the striated muscle was identified in the rodent esophagus. This reflex circuit consists of primary afferent neurons and myenteric neurons, which can modulate the release of neurotransmitters from vagal motor neurons in the striated muscle esophagus. The pathogenesis of some esophageal disorders such as achalasia and gastroesophageal reflux disease might be involved in dysfunction of the neural networks including alterations of the myenteric neurons. These evidences indicate the physiological and pathological significance of intrinsic nervous system in the regulation of the esophageal motility. In addition, it is assumed that the components of intrinsic neurons might be therapeutic targets for several esophageal diseases.     

Striated myogenesis and peristalsis in the fetal murine esophagus occur without cell migration or interstitial cells of Cajal

Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal.     

Extension of bladder-based organ regeneration platform for tissue engineering of esophagus

Recent successes in regenerative medicine and tissue engineering of bladder and bladder-like neo-organs have leveraged regenerative constructs composed of a biodegradable scaffold seeded with a population of smooth muscle cells. We have shown that such smooth muscle cells are isolatable from adipose and other sources alternate to the primary organ. We hypothesize that this regenerative platform is not limited to regeneration of bladder and bladder-like neo-organs, but rather represents a foundational technology platform broadly applicable for regeneration of laminarly organized hollow organs. Using esophagus as an illustrative example in support of this hypothesis, we demonstrate that patch constructs composed of adipose-derived smooth muscle cells seeded on a biodegradable matrix catalyze complete regeneration of the esophageal wall in a rodent model of esophageal injury. By implication, such regenerative constructs may potentially be used to mediate the regeneration of any laminarly organized tubular organ.     

Both smooth and skeletal muscle precursors are present in foetal mouse oesophagus and they follow different differentiation pathways.

Muscularis externa of mouse oesophagus is composed of two skeletal muscle layers in the adult. Unlike rest of skeletal muscle in the body, the oesophageal skeletal muscle in the mouse has been proposed to be derived from fully differentiated smooth muscle cells by transdifferentiation during later foetal and early postnatal development (Patapoutian et al. [1995] Science 270:1818-1821). Here we characterised the nature of cells in muscularis externa of the mouse oesophagus by ultrastructural and immunoctyochemical analyses. The presence of differentiated skeletal muscle cells identified by positive staining for skeletal muscle specific myosin heavy chain became first apparent in the outer layer of cranial oesophagus at 14 days gestation. The transient expression of smooth muscle type alpha-actin in mouse oesophageal muscle was also apparent during foetal development. This isoform, however, was not smooth muscle specific during early development as it was also detected in foetal skeletal muscles. Compared with oesophagus, the suppression of this smooth muscle type alpha-actin during foetal development was faster in non-oesophageal skeletal muscle cells. The development of skeletal muscle in oesophagus showed a cranial to caudal and an outer layer to inner layer progression. During early foetal development, mouse oesophagus is composed of undifferentiated mesenchymal cells that formed cell clusters. Two types of cells with different staining densities could be distinguished within these cell clusters by electron microscopy. The centrally located pale staining cells gave rise to skeletal muscle cells while the peripherally positioned dense staining cells gave rise to smooth muscle cells, indicating the existence of both skeletal and smooth muscle cell precursors in mouse oesophagus during early foetal development. Further development showed an increase in the proportion of skeletal muscle cells and a decrease in size and number of the smooth muscle type cells. Apart from decrease in cell size, some other morphological features of smooth muscle cell degeneration were also observed during later foetal and early neonatal development. No smooth muscle cells undergoing transdifferentiation were observed. Both immunochemical and ultrastructural observations, thus, demonstrated the presence of skeletal muscle cells in early foetal oesophagus. It is concluded that the transient appearance of smooth muscle cells may provide a scaffold for the laying down of skeletal muscle layers in mouse oesophagus, the final disappearance of which may be triggered by lack of smooth muscle innervation.     

Human smooth muscle myosin heavy chain gene mapped to chromosomal region 16q12

The partial nucleotide sequence encoding the rod portion of the entire amino acid sequence of human smooth muscle myosin heavy chain (MHC) which corresponds to MYH11, according to Human Gene Mapping nomenclature, has been determined by cloning a complementary DNA (cDNA) and sequencing the cDNA (UMYHSM). Northen blot analysis with the UMYHSM fragment (4.3 Kb) showed that the smooth muscle MHC of the human umbilical artery is expressed in the human umbilical artery, bladder, esophagus and trachea. Southern blot analysis of human genomic DNA from human-mouse or human-Chinese hamster somatic cell hybrids demonstrated that the human smooth muscle MHC was mapped to human chromosome 16. Regional mapping of UMYHSM was performed using human cell lines with partial deletion and trisomy of chromosome 16. As a result, the human smooth muscle MHC gene segregated with 16p11-q12. In situ hybridization of biotin-labeled human smooth muscle MHC probe (UMYHSM fragment) to normal human metaphase chromosome independently showed that the human smooth muscle MHC gene (MYH11) is assigned to chromosome region 16q12. Analysis of early metaphase chromosomes showed that hybridization signals were in 16q12.1. In the human, although skeletal, cardiac, smooth muscle, and nonmuscle MHC genes are mapped to chromosomes 17, 14, 16 and 22, respectively, structural similarities of these MHC genes strongly suggest the common origin of these genes. © 1993 Wiley-Liss, Inc.     

Neuronal substance P in the esophagus. Distribution and effects on motor activity

Substance P-immunoreactive nerve fibres were fairly numerous in the lower esophagus of the guinea-pig and cat but few in the pig. They were particularly numerous in the myenteric and submucosal plexuses but could be detected also in the circular and longitudinal smooth muscle and in the muscularis mucosae. Only in the cat were SP-immunoreactive cell bodies detected, albeit in low number, in the myenteric plexus. Radioimmunoassay showed that the lower part of the cat esophagus contained approximately 10 times more immunoreactive SP than the upper part and that the muscle layer contained more SP than the mucosa. Motor effects of synthetic SP were studied on segments from circular smooth muscle of cat esophagus. SP contracted the smooth muscle and enhanced the response to electrical stimulation. These effects of SP could be blocked by the specific SP antagonist (d -Pro,²d -Trp^7,8)-SP. The contractile response to electrical stimulation could be blocked by the cholinergic muscarinic blocker atropine and the opiate receptor agonist leu-enkephalin but not by the SP antagonist or by adrenergic blockers. Hence, the results suggest that cholinergic neurons innervate the circular smooth muscle, and that opiate receptor agonists suppress transmission in these neurons. Neuronal SP in the esophagus may serve to enhance the contractile responses of esophageal smooth muscle.     

Distribution of vagal CGRP-immunoreactive fibers in the lower esophagus and the cardia of the stomach of the rat

We have examined whether the smooth muscle fibers in the lower esophagus and the cardia of the stomach of the rat are innervated by calcitonin gene-related peptide-immunoreactive (CGRP-ir) fibers coming from the nucleus ambiguus. Immunohistochemical observations revealed that there were many CGRP-ir fibers and free endings in all external muscular layers of the lower esophagus and the cardia. Occasionally, bundles of CGRP-ir fibers were found in the inner oblique muscle layer of the cardia. There were also many CGRP-ir fibers in the mucous membrane in the lower esophagus and the cardia. When Fluorogold was injected into the junction of the lower esophagus and the cardia, many retrogradely labeled neurons were found in the compact formation of the nucleus ambiguus and the dorsal motor nucleus of the vagus nerve. Double labeling with immunohistochemistry for CGRP and the retrograde tracer Fluorogold showed that almost all of neurons (more than 90%) in the nucleus ambiguus that project to the lower esophagus or the cardia contained CGRP, while no CGRP-ir neurons were found in the dorsal motor nucleus of the vagus nerve. These results indicate that the vagal motor neurons of the nucleus ambiguus that contain CGRP project not only to the striated muscle fibers of the esophagus but also to the smooth muscle fibers of the external muscle layers of the lower esophagus and the cardia.     

Juvenile megaesophagus in PKCα-deficient mice is associated with an increase in the segment of the distal esophagus lined by smooth muscle cells

Megaesophagus in mice has been associated with several genetic defects. In the present study we expand the range of genes associated with esophageal function and morphology by protein kinase C alpha (PKCα). PKCα-deficient mice showed a six times increased prevalence of megaesophagus at the age of 9–10 weeks compared to wild-type animals. In contrast, in a restricted number of 14-month-old animals of both genotypes a similar prevalence of megaesophagus was found. Megaesophagus was associated with an increased portion of the distal esophagus lined by smooth muscle cells. Achalasia-like degeneration or loss of neuronal cells, inflammation or fibrosis was not present in any of the animals. The results of the study therefore suggest that PKCα expression is associated with a delayed replacement of embryonic smooth muscle by skeletal muscle at the distal esophagus and consecutive megaesophagus in young mice, which, however, is not present at the same prevalence at an advanced age.     

The organization of the lamina muscularis mucosae in the human esophagus

The structural organization of the lamina muscularis mucosae of the human esophagus was studied by light microscopy and scanning electron microscopy (SEM). The organization of the lamina muscularis mucosae varied considerably among the cervical, the thoracic, and the abdominal part of the esophagus. In the cervical part, the lamina muscularis mucosae was not well developed and only islets of the smooth muscle bundles were scattered within the connective tissue. In the thoracic part, the lamina muscularis mucosae consisted of several layers of smooth muscle bundles, individual muscle cells of which ran in a longitudinal direction. In the abdominal esophagus near the cardia, the muscular bundles in the lamina muscularis mucosae ran in various directions forming a reticular configuration. The differences in density and arrangement of the lamina muscularis mucosae are discussed in relation to the swallowing of food and submucosal invasion of esophageal cancer.     

New aspects of vascular remodelling: the involvement of all vascular cell types

Conventionally, the architecture of arteries is based around the close-packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self-maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re-arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.     

A lytic monoclonal antibody to Trypanosoma cruzi bloodstream trypomastigotes which recognizes an epitope expressed in tissues affected in Chagas' disease.

It has been suggested that molecular mimicry between the antigens of Trypanosoma cruzi and the host could have a role in the onset of the chronic stage of Chagas' disease. In this article, we report on a monoclonal antibody (MAb), CAK20.12 (immunoglobulin G2b), which reacts with a polypeptidic epitope of a 150-kDa antigen expressed on the surface of several strains of T. cruzi. This MAb also causes lysis of bloodstream trypomastigotes. Serum samples from 30 of 30 patients with chronic and 11 of 13 patients with acute Chagas' disease present specific antibodies to this antigen. MAb CAK20.12 reacts, by indirect immunofluorescence, with human and syngeneic murine striated muscle tissue, with the smooth muscle layer of cardiac arteries, with the lamina muscularis mucosae and the external striated muscle layer of the esophagus, and with the smooth muscle cells of the colon from normal syngeneic mice. Reactivity with the small intestine was very weak, and no reactivity with ventricle or atrium tissue was detected. Adsorption with an antigenic fraction from normal murine striated muscle or from T. cruzi epimastigotes confirmed that MAb CAK20.12 recognizes a common epitope present in parasites and host tissues. MAb CAK20.12, lytic for the infective form of T. cruzi, recognizes an epitope expressed in striated and smooth muscle cells of the host tissues affected in the chronic stage of Chagas' disease.     

Human atherosclerosis. IV. Immunocytochemical analysis of cell activation and proliferation in lesions of young adults.

The accumulation of smooth muscle cells is a major phenomenon associated with the pathogenesis of lesions of atherosclerosis. Smooth muscle cell proliferation in response to the release of growth factors from neighboring cells, both smooth muscle and macrophages, is one mechanism postulated to account for the increasing numbers of smooth muscle cells as atherosclerotic lesions progress. Indeed, we recently demonstrated the B chain of platelet-derived growth factor (PDGF-B), a potent smooth muscle mitogen, within macrophages in monkey and human lesions of atherosclerosis. To further test the hypothesis that smooth muscle proliferation and/or activation (eg, expression of major histocompatibility complex proteins) plays a role in the early development of these lesions, we applied antibodies to PDGF-B, HLA-DR (a marker of cell activation), and proliferating-associated marker) on a series of early human atherosclerotic lesions from young adults in conjunction with cell-type-specific antibodies. Smooth muscle cells had previously been demonstrated to comprise a major fraction of the cell population in these lesions. In a continuing study of early and intermediate lesions of individuals ranging in age from 15 to 34 years, PDGF-B was detected within macrophages in 2 of 15 lesions. There was no evidence of HLA-DR expression by the smooth muscle cell population in any of the lesions. PCNA-positive cells comprised less than 2% of the cells in the lesions, and the majority of these were blood-borne cells (macrophages and/or lymphocytes), although a small fraction of the PCNA-positive cells were identified as smooth muscle. Concurrent PCNA and 5'-bromodeoxyuridine studies of peripheral blood monocytes demonstrated the presence of significant numbers of cells positive for these proliferation-related markers. It is concluded that the growth factor PDGF-B may have a role in regulating cell proliferation in early human fatty streaks, but the number of proliferating smooth muscle cells is relatively small, and there is no evidence of smooth muscle cell activation, as judged by HLA-DR positivity, in these lesions.