Heterogeneous expression of nestin in myofibroblasts of various human tissues

In this study we explored the distribution of nestin‐positive cells in extraneural human tissues with special reference to stromal myofibroblasts. Tissue microarrays were constructed from various tissues with normal histology and tissues with fibrosing disorders. Sections were immunostained for nestin, alpha‐smooth muscle actin (alpha‐SMA), desmin, vimentin, CD34, and other stromal markers. Nestin was expressed in the myoepithelium of the breast, podocytes of the renal glomerulus, and endothelial cells of most organs. Nestin was also expressed in the stroma of several organs, including the intestine, uterine cervix, and endometrium. Nestin‐positive fibroblast‐like cells appeared in the stroma of the kidney, pancreas, lung, and skin in fibrosing conditions. With the notable exception of endometrial stromal cells, most of these nestin‐positive stromal cells were alpha‐SMA‐positive. Interestingly, we observed a concomitant appearance of nestin‐ and CD34‐positive myofibroblasts under fibrosing conditions. Further investigation showed that nestin was expressed by stromal fibroblasts in cervical squamous cell carcinoma, but not in lung adenocarcinoma, pointing to heterogeneity of cancer stroma. In conclusion, nestin was expressed in variable proportions of stromal myofibroblasts in human tissues. The differential expression of nestin may indicate phenotypic and functional heterogeneity. Nestin‐positive myofibroblast may represent a relatively immature subpopulation of cells with multipotentiality.     

Re-expression of nestin in the myocardium of postinfarcted patients

Intact cardiac muscle cells in the adult heart do not express intermediate filament nestin. In this study, we report on widespread expression of intermediate filament nestin in human myocardium of patients who died from the myocardial infarction. Nestin was detected in cardiomyocytes, endothelial cells, and few interstitial cells. Elevated levels of nestin were observed in cardiac muscle cells in all specimens, although the intensity of immunoreactivity and distribution of the signal differed. The strongest immunoreactivity was observed from 4 days after myocardial infarction in the infarction border zone where nestin was distributed homogeneously in the entire sarcoplasm of cardiac muscle cells. Within the following week, nestin in immunoreactive cardiomyocytes was redistributed and restricted to small subsarcolemmal foci and to intercalated discs. Angiogenic capillaries that grew between vital nestin-positive cardiomyocytes and entered the necrotic area expressed also high levels of nestin. Nestin-positive endothelial cells were often observed in mutual interactions with nestin-positive cardiac muscle cells. These findings document a crucial role of nestin in remodeling cytoskeleton of cells in the human postinfarcted myocardium.     

Nestin expression in odontoblasts and odontogenic ectomesenchymal tissue of odontogenic tumours

BACKGROUND: Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine. AIMS: The aim of this study was to investigate nestin expression in various odontogenic tumours and evaluate its usefulness for histopathological diagnosis. METHODS: We studied formalin fixed, paraffin embedded specimens from 129 cases of odontogenic tumours and 9 of mandibular intraosseous myxoma. After characterisation of odontogenic ectomesenchymal tissues in these tumours using antibodies to vimentin, desmin, neurofilament, and glial fibrillary acidic protein, we immunohistochemically examined nestin expression. RESULTS: No differentiation towards muscle and nervous tissues was found in the odontogenic ectomesenchymal tissues. Although almost all the ameloblastomas and malignant ameloblastomas were negative for nestin, odontogenic ectomesenchyme in the odontogenic mixed tumours demonstrated nestin immunolocalisation, particularly in the region adjacent to the odontogenic epithelium. Odontoblasts and their processes, pulp cells near the positive odontoblasts, and flat cells adhering to the dentine showed immunoreaction with nestin in the odontomas and odontoma-like component in the ameloblastic fibro-odontomas. Neoplastic cells in almost half cases of jaw myxoma and one case of odontogenic fibroma expressed nestin. CONCLUSIONS: The distribution of nestin in the odontogenic mixed tumours suggests that nestin expression in the odontogenic ectomesenchyme is upregulated by stimulation from odontogenic epithelium. In addition, nestin may also be involved in the differentiation from pulp cells to odontoblasts in odontogenic tumours. Therefore, nestin is a useful marker for the odontogenic ectomesenchyme and odontoblasts in odontogenic tumours. Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine.     

Expression of nestin in rat and human glomerular podocytes

Nestin is a neuroepithelial precursor cell marker expressed in a variety of human cell types during development. However, no information exists on the expression of nestin in mature glomeruli as well as during the glomerular development. Here, we examined nestin expression in rat and human glomerular tissues in quiescent states using RT-PCR and immunohistochemical methods. Nestin mRNA was detected in the rat glomeruli in parallel with its expression in developing rat brains. In the normal mature rat glomeruli, WT-1 positive cells expressed nestin. Co-expression of nestin and vimentin was observed in mature rat podocytes. Immunoelectron microscopy revealed nestin localization in the cell bodies and primary processes of podocytes. A similar expression pattern was observed for vimentin. In matured glomeruli, nestin was not expressed by mesangial and endothelial cells. In the newborn rat, early developing glomeruli (metanephric cap, metanephric vesicle, comma-shaped vesicle and S-shaped body phases) expressed nestin. In the capillary loop stage, Bowman's capsules also expressed nestin. Immunoelectron microscopy demonstrated that developing podocytes and endothelial cells in S-shaped phase glomeruli expressed nestin. Additionally, in immature glomeruli, the mesangial cells in capillary stage of glomerulus also expressed nexin. As in the rat, WT-1 positive cells in human glomeruli also expressed nestin and immunoelectron microscopy confirmed nestin expression in human glomerular podocytes. These results reveal that in normal condition nestin is expressed in several glomerular cell types at early stage of development and becomes confined to podocytes in mature glomeruli, thus implicating nestin in podocyte functions.     

Altered distribution of desmin filaments in hypertrophic cardiomyopathy: an immunohistochemical study.

Hypertrophic cardiomyopathy (HCM) is characterized by myofiber hypertrophy and disarray. Intermediate filaments play an important role in maintaining of normal cell shape. Desmin filaments have been detected in adult cardiomyocytes, and vimentin and keratin filaments in cardiomyocytes during embryonic development. The pattern of arrangement of intermediate filaments in HCM has not been reported. We examined the distribution of intermediate filaments in formalin-fixed tissue sections of the disarrayed myofibers from 10 hearts with HCM using an immunohistochemical technique and antibodies to desmin, vimentin, and high and low molecular weight keratins. The controls consisted of subaortic tissue from surgically explanted hearts of patients with ischemic heart disease. In the ischemic hearts, desmin was detected in the Z bands and intercalated disks. In all HCM cases, three patterns of staining for desmin were noted: (a) individual myocytes showing a parallel arrangement along Z bands; (b) focal myofibers with decreased or complete loss of labeling of Z bands; and (c) individual myocytes with intense granular cytoplasmic staining especially in disarrayed myofibers. No staining for vimentin or keratins was noted in the cardiomyocytes from either the HCM or ischemic cases. The altered arrangement of desmin filaments in the disarrayed cardiac muscle fibers may play a role in the altered contractility that occurs in patients with HCM.     

Expression of nestin after renal transplantation in the rat

Chronic allograft injury (CAI) limits the long‐term success of renal transplantation. Nestin is a marker of progenitor cells, which probably contribute to its pathogenesis. We hypothesize that nestin is induced by ischemia/reperfusion injury and acute rejection, main risk factors for CAI. Syngeneic renal transplantation was performed in Lewis rats and allogeneic transplantation in the Fischer 344 to Lewis strain combination, which results in reversible acute rejection and in CAI in the long‐run. The Dark Agouti to Lewis rat strain combination was used to study fatal acute rejection. In untreated kidneys, nestin immunoreactivity was detected in glomeruli and in very few interstitial or microvascular cells. Syngeneic transplantation induced nestin expression within 4 days, which decreased until day 9 and returned to control levels on day 42. Nestin expression was strong during acute rejection and still detected during the pathogenesis of CAI on day 42. Nestin‐positive cells were identified as endothelial cells and interstitial fibroblast‐like cells co‐expressing alpha‐smooth muscle actin. A sub‐population of them expressed proliferating cell nuclear antigen. In conclusion, nestin is induced in renal grafts by ischemia/reperfusion injury and acute rejection. It is expressed by proliferating myofibroblasts and endothelial cells and probably contributes to the pathogenesis of CAI.     

Nestin expression reflects formation, revascularization and reinnervation of new myofibers in regenerating rat hind limb skeletal muscles

This work provides a comprehensive view of expression and distribution patterns of the intermediate filament nestin in rat regenerating skeletal muscles. Regeneration was induced by heterochronous isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. Recipients were sacrificed by anesthetic overdose and the host muscles with the graft were excised after 7-, 16-, 21- and 29-day survival, fixed in 10% formalin solution and immunohistochemically stained. In the regenerating skeletal muscle maximal nestin expression was observed in newly formed myoblasts and myotubes and it decreased in the course of skeletal myofiber development. We demonstrate the phenomenon of a peripheral concentration of nestin expression within the differentiating myofibers called 'lateralization', which may represent a mechanism of nestin elimination during the development of skeletal myofibers. Furthermore, nestin has been detected in newly formed endothelial cells of blood vessels growing into the regenerated area and also in peripheral nerves innervating the graft as well as the host muscle. In conclusion, according to our results, nestin represents an ideal marker for skeletal muscle regeneration, as its expression reflects processes of development, revascularization and reinnervation of new myofibers.     

Involvement of cytoskelatal proteins and growth factor receptors during development of the human eye

The spatial and temporal distribution of nestin, cytokeratins (CKs), vimentin, glial fibrillary acidic protein (GFAP), neurofilaments (NFs), β-tubulin as well as fibroblast growth factor receptors (FGFRs) and platelet-derived growth factor receptor β (PDGF-Rβ) were investigated in the developing human eye in eight conceptuses of 5–9 postovulatory weeks using immunostaining. Nestin was found in the neuroglial precursors and the radial glial fibres of the optic nerve. In the pigmented retina, nestin was present only in the 5th week, while at later stages (6–9th week), co-expression of CKs and vimentin was seen. Nestin, CKs, vimentin, and GFAP were observed in the precursors to various cell types in the neural retina. Additionally, their expression was also apparent in the lens epithelium, showing its gradual fading following the lens fibre elongation. They appeared in the mesenchymal cells of the cornea, the choroid, the sclera, and the corpus vitreum, too. In the corneal epithelium, co-expression of nestin and CKs was detected. NFs and β-tubulin were confined to the differentiating retinal neuroblasts. Growth factor receptors were seen in the retina, the lens epithelium while less intensely in the lens fibres, the corneal epithelium, and the mesenchymal cells. During the early eye development (5–9th week), IFs expressing normal pattern of distribution as well as acting in concert might contribute to the normal developmental processes occurring in certain parts of the human eye. Additionally, NFs and β-tubulin seem to have an important role in the retinal ganglion cell differentiation, while FGFRs and PDGF-Rβ may regulate the cell proliferation, differentiation, and survival in various parts of the developing eye.     

Intermediate filament proteins increase during chronic stimulation of skeletal muscle

Summary Chronic low-frequency electrical stimulation of rabbit fast-twitch skeletal muscle induces increased levels of two intermediate filament proteins, desmin and vimentin, during the first 3 weeks of stimulation. These increases occur over the same timecourse as reported shifts in -actinin expression and increased Z-disc width, but precede the fast-to-slow shifts in contractile proteins, which have been described by others. Desmin and vimentin levels increase during the first 2 weeks of stimulation, at which time the increase in desmin appears to plateau while vimentin continues to increase significantly through 3 weeks of stimulation. Absolute amounts of vimentin are lower than desmin at all time points, however increases in desmin and vimentin levels are strongly correlated during the stimulation period, suggesting that the two proteins are coordinately increased during the initial phases of muscle transformation. We suggest that rapid increases in the expression of intermediate filament proteins, which coincide with alterations in Z-disc structure, may indicate a fortification of the force-bearing ultrastructure of the muscle fibre in response to the increased activity that is induced by stimulation. The presence of vimentin and elevated levels of desmin expression suggest that mature skeletal muscle reverts toward a developmental program of intermediate filament protein expression during fast-to-slow transformation.     

Involvement of cytoskeletal proteins and growth factor receptors during development of the human eye

The spatial and temporal distribution of nestin, cytokeratins (CKs), vimentin, glial fibrillary acidic protein (GFAP), neurofilaments (NFs), β-tubulin as well as fibroblast growth factor receptors (FGFRs) and platelet-derived growth factor receptor β (PDGF-Rβ) were investigated in the developing human eye in eight conceptuses of 5–9 postovulatory weeks using immunostaining. Nestin was found in the neuroglial precursors and the radial glial fibres of the optic nerve. In the pigmented retina, nestin was present only in the 5th week, while at later stages (6–9th week), co-expression of CKs and vimentin was seen. Nestin, CKs, vimentin, and GFAP were observed in the precursors to various cell types in the neural retina. Additionally, their expression was also apparent in the lens epithelium, showing its gradual fading following the lens fibre elongation. They appeared in the mesenchymal cells of the cornea, the choroid, the sclera, and the corpus vitreum, too. In the corneal epithelium, co-expression of nestin and CKs was detected. NFs and β-tubulin were confined to the differentiating retinal neuroblasts. Growth factor receptors were seen in the retina, the lens epithelium while less intensely in the lens fibres, the corneal epithelium, and the mesenchymal cells. During the early eye development (5–9th week), IFs expressing normal pattern of distribution as well as acting in concert might contribute to the normal developmental processes occurring in certain parts of the human eye. Additionally, NFs and β-tubulin seem to have an important role in the retinal ganglion cell differentiation, while FGFRs and PDGF-Rβ may regulate the cell proliferation, differentiation, and survival in various parts of the developing eye.     

Nestin, a neuroectodermal stem cell marker, is expressed by bovine sertoli cells

Nestin, an intermediate filament protein is expressed by neuroectodermal stem cells and tumors originating from cells of neuroectodermal and mesenchymal lineages. Nestin expression is prominent in embryos and remains upregulated until 3–6?weeks after birth but is downregulated afterward. Sertoli cells are nucleated somatic cells that are spanned in the seminiferous epithelium and play a critical role in supporting and controlling germ-cell development. In this context, we employed immunocytochemical, Western blot, and Flow cytometric analyses to demonstrate nestin expression in bovine sertoli cells. Immunostaining clearly showed that setoli cells express high levels of nestin, a result which was confirmed by Western blot analysis of purified cells. Intracellular staining of sertoli cells by flow cytometry revealed that around 74% of the cells express this marker. Given the high expression of vimentin by sertoli cells, it is proposed that the expression of nestin in these cells might be required for the formation of stable vimentin/nestin intermediate filament network. In light of these findings, it seems that sertoli cells of mature bull have potentiality of proliferation.     

Myoid gonadal stromal tumor with epithelial differentiation (? testicular myoepithelioma).

A 46-year-old man presented with a cytologically bland testicular tumor composed of spindle cells that showed both epitheliallike (ie, true desmosomes and tonofilamentlike structures) and myogenous differentiation (ie, thin filaments with focal densities and alpha-smooth muscle actin immunoreactivity). Tumor cells were immunoreactive for vimentin and S-100 protein but negative for cytokeratin and desmin. Peritubular myoid cells are present in the normal testis; contain subplasmalemmal micropinocytotic vesicles; show thin filaments with focal densities; and are reactive with desmin, vimentin, and alpha-smooth muscle actin. They have no desmosomes and lie outside the basement membrane of the seminiferous tubules; thus they are not true myoepithelial cells (a cell type not present in the testis). Paradoxically, the current tumor appeared to show bidirectional differentiation, mimicking both a peritubular myoid spindle cell and an epitheliallike cell (possibly similar to the granulosa cell or rete testis epithelial cell). Although the findings suggest myoepithelial differentiation, the cytogenesis of this tumor remains uncertain.     

Nestin expression in repopulating mesangial cells promotes their proliferation

We investigated whether the intermediate filament protein and neural stem cell marker nestin characterizes the glomerular progenitor/reserve cell population immigrating the glomerulus after mesangial cell (MC) injury in the rat (anti-Thy1 nephritis). Nestin expression was investigated by immunohistochemistry and real-time PCR during anti-Thy1 nephritis. Migration and proliferation assays were used to characterize the function of nestin in isolated MCs after nestin knockdown by siRNA. After MC injury during anti-Thy1 nephritis, glomerular nestin was transiently increased during the repopulation phase. At the peak of mesangial proliferation and expansion (day 5) most OX-7-positive MCs expressed nestin largely colocalizing with the activation marker alpha-smooth muscle actin and the proliferation marker PCNA. In contrast to a healthy, non-injured mesangium in vivo, MCs in culture are considered to be in an 'activated, injured state' and express nestin in a generalized distribution with condensed localization around the nucleus as well as intensive staining of cell protrusions such as filopodia. During cell cycle, the percentage of MCs with high nestin levels was increased during S- aupnd G2-phase. Blocking of nestin using specific siRNA resulted in inhibition of cell proliferation but not cell migration. In conclusion, nestin is constitutively expressed in podocytes, but is a marker for repopulating MCs after experimental MC injury in vivo. Nestin promotes MC proliferation in vitro, suggesting a supporting role for nestin during repair reaction.     

Expression of developmentally regulated muscle proteins in rhabdomyosarcomas.

Human skeletal muscle differentiation and maturation follows a precise sequence of events. To investigate whether and to what extent rhabdomyosarcoma (RMS) cells follow a comparable sequence, 29 fresh frozen specimens of RMS (14 primary and 15 relapses) were immunostained with antibodies directed against developmentally regulated myosin heavy chains (MHC), ie, fetal, fast, and slow MHC, in addition to desmin and vimentin. Four distinct patterns of expression were observed: I) RMS cells expressing exclusively vimentin and desmin (n = 7), II) in addition to expression of vimentin and desmin, a minority of neoplastic cells were immunoreactive with fetal MHC (n = 6), III) in addition to pattern II, fast MHC was expressed (n = 7), and IV) RMS cells simultaneously expressing vimentin, desmin, fetal, fast, and slow MHC (n = 9). Accordingly, the proportion of the MHC immunoreactive RMS cells increased gradually along with the four patterns of expression evolving from less than 25% up to 75% for fetal MHC, from less than 25% up to 50% for fast MHC, and up to 25% for slow MHC in the last category. Vimentin and desmin were coexpressed by almost all RMS cells. Double immunostaining revealed that comparable with the myogenic cells in the developing fetal skeletal muscle, expression of fetal MHC could be demonstrated in the same neoplastic cells either in conjunction with fast or slow MHC. In contrast, only in RMS, slow MHC expression in conjunction with fast MHC could be observed in the neoplastic cells. Neither the shape or size of neoplastic RMS cells, nor the histopathological types, nor tumor localization were related to the expression pattern of developmentally regulated MHC (fetal, fast, and slow MHC). These results confirm the commitment of the RMS cells to the myogenic pathway and demonstrate a restricted and aberrant differentiation pattern of the neoplastic cells in RMS compared with normal myogenesis, independent of histopathological types of RMS.     

Relationship between utrophin and regenerating muscle fibers in duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a dystrophinopathy, and its associated gene is located on Xp21. Moreover, utrophin, a recently identified structural homologue of dystrophin is reported to be up-regulated in DMD. In order to investigate the association between utrophin and muscle regeneration in DMD, an immunohistochemical study using antibodies to utrophin, dystrophin, vimentin and desmin was carried out in 17 cases of DMD, 3 cases of polymyositis and 1 case of dermatomyositis. Dystrophin was negative in almost all cases of DMD, but positive in all cases of inflammatory myopathy (IM). Utrophin was positive in 94.0% of DMD and in 75.0% of IM. 36.4% of the myofibers were positive in DMD, as compared to 10.5% in IM (p=0.001). In both groups, utrophin positivity was present most commonly in small regenerating fibers (p=0.001, 0.013). Vimentin and desmin were intensely positive in regenerating fibers in all cases of DMD and IM. 34.4% and 35.4% of myofibers were positive for vimentin and desmin in DMD, as compared to 21.8% and 20.9% in IM (p=0.001, 0.001). In both groups, vimentin and desmin positivity were present most commonly in small regenerating fibers (p=0.001, 0.001). The staining intensities of utrophin, vimentin and desmin were also higher in small regenerating fibers. These results show that utrophin up-regulation is regeneration-associated, and that it is proportional to the quantity of regenerating myofibers, but is not specific for DMD.     

巢蛋白在人胚胎消化管的表达

目的　通过研究不同时期人胚胎消化管巢蛋白的表达及其变化探讨巢蛋白在消化管发育过程中的作用。 方法　取3~8月人胚胎食管、胃和小肠,常规石蜡切片,采用免疫组织化学法检测巢蛋白在胚胎消化管中的表达及分布。 结果　各胎龄段（3~8月）消化管均有巢蛋白的表达。巢蛋白在消化管主要表达在固有层、黏膜下层和肌层内的微血管和毛细血管壁上,且巢蛋白的表达随胎龄的增加而减少。与发育早期组相比,发育中期、晚期胚胎食管、胃和小肠组织中巢蛋白的表达显著减少（P<0.05）,差异有统计学意义。而在发育中期和发育晚期巢蛋白的表达无显著性差异（P>0.05）。 结论　巢蛋白在胎儿消化管壁的微血管和毛细血管的管壁上表达,且随着胎儿发育,其表达逐渐减少,提示巢蛋白在这些器官组织的血管形成过程中起作用。     

Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins

We examined by immunofluorescence the distribution of vimentin, desmin, alpha-smooth muscle actin and alpha-sarcomeric actin in normal human soft tissues and in pathologic tissues containing myofibroblasts, including normally healing granulation tissue, hypertrophic scar, and fibromatosis. The pattern of actin isoforms was also documented biochemically by two-dimensional gel electrophoresis. Fibroblastic and/or myofibroblastic cells in each setting always expressed vimentin and never alpha-sarcomeric actin. Moreover, these cells showed an heterogeneous cytoskeletal composition which defined four phenotypes: (a) cells expressing only vimentin; (b) cells expressing vimentin, alpha-smooth muscle actin and desmin; (c) cells expressing vimentin and alpha-smooth muscle actin; and (d) cells expressing vimentin and desmin. Given this, two groups of lesions are distinguished: the first contains only vimentin cells and consists of normally healing granulation tissue, eschars and normally healed scars; the second contains vimentin cells admixed with variable proportions of vimentin, alpha-smooth muscle actin and desmin, vimentin and alpha-smooth muscle actin, and vimentin and desmin cells and consists of hypertrophic scars and fibromatoses. Immunogold electron microscopy showed that alpha-smooth muscle actin was present in a proportion of cells with ultrastructural features of myofibroblasts. Our findings suggest that contrary to myofibroblasts of normally healing granulation tissue and normally healed scars, myofibroblasts of pathologic conditions characterized by chronic retraction express always immunochemical features indicative of smooth muscle differentiation.     

Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal

It has recently been proposed that gastrointestinal stromal tumors (GISTs) originate from stem cells that differentiate toward a phenotype of interstitial cells of Cajal (ICCs). Nestin is a newly identified intermediate filament protein, and is predominantly expressed in immature cells, such as neuroectodermal stem cells and skeletal muscle progenitor cells, and tumors originating from these cells. In this study, we examined, using immunohistochemistry, the nestin expression in GISTs and ICCs to clarify the origin of GISTs. Strong immunoreactivity for nestin was observed in all 18 GISTs, and its expression was confirmed by Western blot and Northern blot analyses. In contrast, three leiomyomas and a schwannoma that developed in the gastrointestinal tract showed no apparent immunoreactivity for nestin. Among 17 mesenchymal tumors (seven leiomyosarcomas, five malignant peripheral nerve sheath tumors, and five fibrosarcomas) that occurred in sites other than the gastrointestinal tract, only two malignant peripheral nerve sheath tumors were moderately immunoreactive for nestin. Furthermore, with fluorescence double immunostaining of the normal small intestine, nestin expression was demonstrated in ICCs. These results show that nestin may be a useful marker for diagnosis of GISTs, and support the current hypothesis that GISTs are tumors of stem cells that differentiate toward an ICC phenotype.