[Growth defects of extremities].
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Animals↗
Biomedical subjects
J Peltonen
Publications and source records attributed to J Peltonen.
[How are nerves supported--structure and function of connective tissue of nerves].
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Cells, Cultured↗
[Neurofibromatosis].
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Genes, Neurofibromatosis 1↗
Combined use of in situ hybridization and unlabeled antibody peroxidase anti-peroxidase methods: simultaneous detection of type I procollagen mRNAs and factor VIII-related antigen epitopes in keloid tissue.
In this study, we developed methodology that allows the combined use of in situ hybridization and peroxidase anti-peroxidase techniques on the same tissue section. A human pro alpha 1(I) collagen cDNA and antibodies to factor VIII-related antigen were used on keloid tissue sections as a model for a fibrotic reaction. The basic protocols of the techniques were modified to obtain optimal results. The feasibility of this new method was demonstrated by elucidation of type I procollagen gene expression in the cells of blood vessel wall and the adjacent fibroblasts. In the case of capillaries, pro alpha 1(I) collagen mRNAs were detected within endothelial cells identified by the presence of factor VIII-related antigen. Pro alpha 1(I) collagen mRNAs were also found in close proximity of medium-size blood vessels, but in this context clearly outside the vessel wall. These results may contribute to the understanding of pathogenetic aspects of keloids and other fibrotic conditions. Thus, the combination of in situ hybridization and peroxidase anti-peroxidase techniques provides a useful tool to examine gene expression simultaneously both at mRNA and protein levels in fibrotic tissues. This methodology is also applicable to a variety of other biologic and pathologic situations.
DNA↗
Presence of type I and VI collagen mRNAs in endothelial cells in cutaneous neurofibromas.
In situ hybridization and peroxidase anti-peroxidase immunodetection were used in the same tissue sections to elucidate the spatial distribution of collagen gene expression in cutaneous neurofibromas, particularly in relation to blood vessels; the latter structures were identified by the presence of factor VIII-related antigen. The data indicate a clear relationship between the vascular structures and sites of locally elevated expression of type I and VI collagen genes. Specifically, some, but not all, blood vessels were surrounded by stromal cells highly active in expressing pro alpha 1(I) and alpha 2(VI) collagen genes. Furthermore, these genes were expressed by a subpopulation of endothelial cells within the walls of blood vessels traversing the lesion. To quantitate the overall expression of five genetically distinct collagen genes in cutaneous neurofibromas, we performed Northern analyses and slot blot hybridizations with pro alpha 1 (I), pro alpha 2 (I), pro alpha 1 (III), pro alpha 1 (IV) and alpha 2(VI) collagen cDNAs. Although the mRNA levels for all five genes were slightly increased in neurofibroma tissue, only the abundance of alpha 2(VI) collagen mRNAs was significantly elevated, as compared with normal skin. We conclude that endothelial cell populations with different levels of collagen gene expression exist within cutaneous neurofibromas: some are actively expressing type I and VI collagen genes, whereas in other the expression of these genes is effectively down-regulated. The markedly elevated steady-state levels of type VI collagen mRNAs suggest that synthesis of type VI collagen may contribute to the growth and architecture of cutaneous neurofibromas.
Collagen↗
Differential modulation of basement membrane gene expression in human fibrosarcoma HT-1080 cells by transforming growth factor-beta 1. Enhanced type IV collagen and fibronectin gene expression correlates with altered culture phenotype of the cells.
The effects of transforming growth factor-beta 1 (TGF-beta 1) on the regulation of basement membrane gene expression were studied in human fibrosarcoma HT-1080 cell cultures. Treatment of cells with TGF-beta 1 resulted in a time- and dose-dependent enhancement of type IV collagen and fibronectin gene expression, as detected both at the mRNA level by Northern hybridizations and at the protein level by semi-quantitative indirect immunofluorescence analyses. These changes were accompanied by profoundly altered morphology of the cell cultures. In contrast, laminin B1 and B2 chain, and nidogen mRNA levels remained unaltered by TGF-beta 1 in the same cultures, indicating uncoordinate modulation of the expression of basement membrane components by TGF-beta 1. Cycloheximide experiments provided evidence that the TGF-beta 1-elicited upregulation of the expression of the fibronectin gene is, but that of type IV collagen is not, entirely dependent on protein synthesis, suggesting two different mechanisms for enhancement of gene expression. Incubation of HT-1080 cells with TGF-beta 1 also resulted in a slightly enhanced expression of beta 1 and alpha 5 integrin mRNAs, as well as beta 1 integrin epitopes. Furthermore, incubation of the cells with anti-beta 1 integrin antibodies partially counteracted the TGF-beta 1-induced morphologic alterations. These studies provide evidence for the role of beta 1 integrins in TGF-beta 1 elicited alterations in HT-1080 cell culture morphology, possibly mediated by the expression of ligand proteins for these integrins, such as type IV collagen and fibronectin.
Basement Membrane↗
Development of diffuse fasciitis with eosinophilia during L-tryptophan treatment: demonstration of elevated type I collagen gene expression in affected tissues. A clinicopathologic study of four patients.
We describe the cases of four women who developed a scleroderma-like syndrome during L-tryptophan treatment for insomnia or tinnitus. The illness was characterized by swelling of the extremities, skin rash, myalgia, and elevation of the peripheral blood eosinophil count, followed by rapidly progressive cutaneous and subcutaneous induration. The histopathologic examination of affected skin showed thickening of the fascia, deep dermal fibrosis, and accumulation of mononuclear cells and abundant eosinophils. The expression of the type I procollagen gene was examined by in-situ hybridizations of affected skin with a human sequence-specific complementary DNA (cDNA). Increased hybridization signals were detected in the deep dermis and fascia, indicating enhanced expression of the collagen gene. The temporal association of L-tryptophan use and the development of a scleroderma-like illness in these four patients suggests a causal relation between L-tryptophan or its metabolites and the stimulation of fibroblast collagen gene expression that results in dermal and fascial fibrosis.
Aged↗
Increased expression of type VI collagen genes in systemic sclerosis.
The expression of type VI collagen genes in affected skin from patients with systemic sclerosis (SSc) was examined by in situ hybridizations with a human alpha 2(VI) collagen sequence-specific complementary DNA. Five patients with diffuse, rapidly progressive SSc of recent onset (less than 12 months) were studied. The results showed increased expression of alpha 2(VI) collagen messenger RNA transcripts in the skin of scleroderma patients compared with that in the skin of normal subjects. These findings indicate that alterations in the expression of type VI collagen genes, similar to those previously described for types I and III collagen, are present in the affected skin of SSc patients. These alterations may result in excessive tissue accumulation of type VI collagen and may play a role in the progressive skin induration and sclerosis that are prominent features of SSc.
Adult↗
Collagen synthesis and mineralization in the early phase of distraction bone healing.
Corticotomy of the distal radius followed by gradual distraction by external fixation was performed on three sheep. Collagen synthesis and mineral deposition were analysed from sequential biopsies obtained from the center of the distraction area during the first 4 weeks of distraction. The whole distraction area was rapidly filled with organic matrix the amount of which, due to fluctuation in its nonprotein component, initially decreased from 88 to 66% of the level in control bone but gained its initial level in 4 weeks. Total protein in the matrix represented 70% of that in the control bone during the 4-week follow up period while the proportion of collagen of the total protein increased from 53 to 88%, a level comparable with the unoperated bone. Determination of the type of fibrillar collagen by characterization of their cyanogen bromide peptides showed that in the distraction area production of type II collagen does not occur but the heteropolymer type I (alpha 1(I)2 alpha 2(I)1) collagen represents almost totally the collagen synthesized. Deposition of mineral into the distraction gap was detectable already after 2 weeks and increased rapidly after 3 weeks of distraction. The results suggest that unlike in other processes, e.g., direct osteonal and callus-type bone repair, in distraction bone healing gradual distraction of osteotomized bone leads directly to synthesis of mature fibrous organic matrix of bone followed by its rapid mineralization.
Animals↗
Segmental neurofibromatosis: immunocytochemical analysis of cutaneous lesions.
Cutaneous lesions from three patients with segmental neurofibromatosis were evaluated. Routine histologic studies revealed the presence of redimentary neural structures within an abundant collagenous matrix. The majority of the cells in all three cases expressed S-100 protein, suggesting their identity as Schwann cells. The stromal component stained positively for fibronectin and type IV collagen; the latter indicated the presence of basement membrane material. Embedded in the tumor mass were glandular epithelial structures that stained with epithelial membrane antigen antibody. Staining for factor VIII-related antigen revealed vascular endothelium and multiple scattered mast cells. In one case strands of cells stained with antibodies to desmin, suggesting muscle cell differentiation. This case may represent a distinct subset of neurofibromas.
Adult↗
Novel function for beta 1 integrins in keratinocyte cell-cell interactions.
We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.
Actins↗
Evaluation of transforming growth factor beta and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization.
Full thickness biopsies of affected skin and fascia from one patient with diffuse fasciitis and eosinophilia (DF), two patients with generalized morphea (GM), and five patients with progressive systemic sclerosis (PSS) of recent onset were examined for the expression of transforming growth factor beta 1 (TGF beta 1) and type I procollagen genes by in situ hybridization with human sequence-specific cDNA. An increased number of fibroblasts showing clearly detectable expression of pro alpha 1(I)collagen gene was found in all fibrotic lesions when compared with unaffected skin from the patient with DF and skin from two normal individuals examined in parallel. Expression of the TGF beta 1 gene was noted in a fibroblast subpopulation of the affected tissues from the patients with DF and GM. In contrast, the TGF beta 1 gene was not expressed at a detectable level in affected skin from the five patients with PSS. The results suggest that TGF beta 1 may play a role in the development of skin fibrosis in cases of DF and GM. However, from these studies, we cannot implicate TGF beta 1 in the pathogenesis of skin fibrosis in PSS.
DNA↗
Distraction effects on the physis in rabbits.
Gradual physeal distraction by external fixation was performed on the distal femur in 19 growing rabbits. The rate of distraction was 0.5-1 mm once daily. Distraction was continued for 1-21 days until killing. Lengthening of the femur and latitudinal growth of the distraction area were measured in radiographs. Morphologic changes in the physes were studied in histologic preparations. The length gained by distraction was maximally 5 mm. Latitudinal overgrowth was noted in specimens distracted for more than 13 days. Hyperplasia of the hypertrophic chondrocyte zone was seen after 1 day of distraction. Separation of the epiphysis from the metaphysis, mostly through the hypertrophic zone, occurred in 11 specimens. Both hyperplasia and separation were often seen in the same physis. In the separation gap, early signs of bone formation into a collagen frame were seen from the 7th day on. An accessory physis, located in the metaphysis and apparently growing interstitially, was noted in 1 specimen.
Animals↗
Type VI collagen. In situ hybridizations and immunohistochemistry reveal abundant mRNA and protein levels in human neurofibroma, schwannoma and normal peripheral nerve tissues.
Cutaneous neurofibromas contain an extensive extracellular matrix composed of collagenous and non-collagenous macromolecules. In this study, the expression of type VI collagen genes in cutaneous neurofibromas was examined by a combination of in situ hybridizations and immunohistochemistry. In situ hybridizations with a 32P-labeled human type VI collagen-specific cDNA revealed that the majority of cells within neurofibromas expressed the gene for alpha 2(VI) collagen chain. The number of cells expressing clearly detectable levels of alpha 2(VI) collagen mRNA was considerably higher than that of cells actively expressing the pro alpha 1(I) or pro alpha 1(III) collagen genes. The presence of type VI collagen epitopes within the neurofibromas was also demonstrated by immunostaining with specific polyclonal antibodies. The expression of type VI collagen genes in neural tissues was further examined by immunostaining of a benign schwannoma tissue specimen consisting of Schwann cells. The results indicated close association of type VI collagen epitopes with the neoplastic Schwann cells. Immunolocalization of type VI collagen epitopes within normal human peripheral nerve revealed pericellular staining of perineurial cells and Schwann cells, suggesting synthesis of type VI collagen by these cell types. These results suggest that the expression of type VI collagen gene is active in nerve-derived tissues, and that type VI collagen may be a major component of the extracellular matrix in neural connective tissues.
Collagen↗
Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes.
Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.
Cells, Cultured↗
Collagen in the extracellular matrix of cultured scleroderma skin fibroblasts: changes related to ascorbic acid-treatment.
The synthesis of collagen and its deposition into the extracellular matrix were studied in scleroderma and normal skin fibroblast cultures using immunocytochemistry, electron microscopy and electrophoretic analysis. Indirect immunofluorescence localized type I collagen to intracellular granules of normal skin fibroblasts but not to the extracellular matrix. A 48-h ascorbic acid-treatment of scleroderma and normal skin fibroblasts induced the appearance of fibrillar, collagen-containing deposits in the extracellular space as indicated by indirect immunofluorescence and electron microscopy. Interestingly, similar type I collagen-containing fibrils were observed in cultures of scleroderma skin fibroblasts even without preceding ascorbic acid treatment. Electrophoretic analysis revealed that ascorbic acid increases the conversion of procollagen to collagen to same extent both in scleroderma and control cell layers. The accumulation of collagen to the cell layer of scleroderma fibroblasts was increased when compared to normal skin fibroblasts; whereas the relative conversion of procollagen to collagen was identical in scleroderma and normal fibroblasts. The results show that the conversion of procollagen to collagen in the cell layers of both scleroderma and normal skin fibroblasts is dependent on ascorbic acid, due to improved secretion of procollagen molecules in the presence of ascorbic acid.
Ascorbic Acid↗
Diabetes induces the formation of large diameter collagen fibrils in the sciatic nerves of BB rats.
Diabetes mellitus (DM) is known to be associated with widespread connective tissue changes. However, the connective tissue of peripheral nerves in diabetes has gained little attention. Thickening of the basement membranes of Schwann, perineurial and endothelial cells suggests a perturbation in the metabolism of type IV collagen. We studied the ultrastructure of endoneurial collagen fibrils in the sciatic nerves of spontaneously diabetic BB rats and found that the fibrils in the diabetic rats were significantly thicker than in the age and sex matched non-diabetic BB rats, both in the proximal (52.6 vs. 46.1 nm) and the distal part (52.4 vs. 45.5 nm) of the nerve, respectively. The mechanism for the thickening of endoneurial collagen fibrils remains unknown, but conceivably involves changes in the biochemical composition of the nerve connective tissue matrix: e.g. altered metabolism of type I and/or type III collagen, increased glycosylation of the collagen molecules, or changes in the glycosaminoglycan content of the ground substance.
Animals↗
Perineurial cells coexpress genes encoding interstitial collagens and basement membrane zone components.
Perineurial cell cultures were established from the sciatic nerves of adult Wistar rats. Highly enriched cultures were studied with respect to the production of extracellular matrix components under conditions free from the influence of Schwann cells, axons, or the extracellular matrix of peripheral nerves. Indirect immunofluorescence staining revealed the presence of collagen type IV epitopes, and electron microscopy demonstrated patches of basement membrane on the perineurial cell surfaces. Collagenous fibrils with a diameter of 15-20 nm were also observed in the intracellular space. SDS-PAGE of radiolabeled medium proteins showed a pattern of bands suggesting the synthesis and secretion of fibronectin, and type I and IV collagens. Northern hybridizations revealed characteristic polymorphic mRNA transcripts corresponding to fibronectin, laminin B2 chain, as well as to the alpha-chain subunits of type I, III, and IV collagens. Furthermore, in situ hybridizations suggested expression of these genes by cultured perineurial cells without apparent heterogeneity within the cell populations. In situ hybridizations of sciatic nerve tissue from 2-wk-old rats also suggested that perineurial cells express alpha 1(I) and alpha 2(IV) collagen, as well as laminin B2 chain genes in vivo. This profile of matrix gene expression is different from that of Schwann cells, which do not synthesize fibronectin, or that of fibroblastic cells, which do not form a cell surface basement membrane. The capability of perineurial cells to express genes for the basement membrane zone and for interstitial collagens further adds to our understanding of the functional role of perineurial cells in developing and healing peripheral nerve, as well as in certain neoplastic lesions of neural origin, such as von Recklinghausen's neurofibromas.
Animals↗