Biomedical subjects
S Sollberg
Publications and source records attributed to S Sollberg.
Scleredema adultorum: case report and demonstration of abnormal expression of extracellular matrix genes in skin fibroblasts in vivo and in vitro.
To elucidate the mechanisms involved in the development of cutaneous fibrosis in scleredema adultorum, we studied a patient with long-standing scleredema who had no history of diabetes mellitus or preceding febrile illness. Histological examination of a biopsy specimen from involved forearm skin demonstrated marked thickening of the dermis and accumulation of mucin between collagen bundles. Increased levels of type I collagen mRNA, as evidenced by positive in situ hybridization signals with an alpha 1(I) procollagen cDNA were found in numerous fibroblasts throughout the dermis. The expression of several genes coding for proteins involved in the maintenance of connective tissue was examined by determining in vitro protein production and mRNA levels in fibroblasts from the affected skin. Total protein production, glucosamine incorporation and collagen synthesis, were elevated by 44-97% in scleredema fibroblasts, compared with fibroblasts from two healthy individuals. Levels of mRNAs for alpha 1(I) and alpha 1(III) procollagens and fibronectin were elevated in scleredema fibroblasts, whereas mRNA levels for the tissue inhibitor of metalloproteinase were unaltered compared with control cultures. The results suggest that fibroblasts from the involved skin in non-diabetic patients with scleredema may exhibit a biosynthetically activated phenotype, which persists for several years. These alterations are likely to be involved in the development of the cutaneous induration and thickening which is characteristic of this disease.
Type VI collagen gene expression in experimental liver fibrosis: quantitation and spatial distribution of mRNAs, and immunodetection of the protein.
Type VI collagen is a minor but essential matrix component in the liver. In this study, we utilized an acute and a chronic injury model to clarify the process of liver fibrosis in rats by administration of carbon tetrachloride. Collagen gene expression, with particular emphasis on type VI collagen, was studied by molecular hybridization techniques. The alpha 2(VI) collagen mRNA levels were markedly elevated on day 3 of acute injury and were approximately at the same high level at 7 and 14 weeks of chronic injury, as determined by Northern hybridizations and slot-blot analyses. Marked enhancement of type I collagen gene expression was similarly noted at these time points. The activation of collagen gene expression in acute injury, as determined by in situ hybridization, was particularly prominent in the vicinity of the central veins. Indirect immunofluorescence demonstrated marked accumulation of type VI collagen protein as early as day 3 of acute injury, and the reaction appeared to be initiated in the proximity of central veins. These results indicate that type VI collagen gene expression, together with other connective tissue components, including type I collagen, is activated in the early stages of the fibrotic process. Type VI collagen accumulation may contribute to the distorted architecture and functional impairment of the liver in hepatic fibrosis.
Sclerosis of the skin in the GEMSS syndrome. An overproduction of normal collagen.
BACKGROUND: We describe a recently observed set of autosomal dominant GEMSS (glaucoma, lens ectopia, microspherophakia, stiffness of the joints, and shortness) syndrome in a 47-year-old woman and her 23-year-old son. In addition, sclerosis of the skin, from which both patients suffered, is investigated in detail. OBSERVATIONS: The histologic examination of skin biopsy specimens obtained from the upper aspects of the backs of both patients revealed a markedly thickened dermis. Immunohistochemical examination of the dermal collagen bundles showed a collagen pattern similar to systemic sclerosis and normal control skin. In situ hybridization showed a markedly enhanced gene expression of transforming growth factor beta 1. CONCLUSION: The sclerotic skin changes in GEMSS syndrome are the result of an abnormally increased production of normal collagen that might be attributable to the enhanced in situ production of transforming growth factor beta 1.
Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses.
Type VII collagen is the predominant, if not the exclusive, component of the anchoring fibrils. In this study, we have examined the expression of the type VII collagen gene in human skin fibroblasts and keratinocytes in culture by Northern analyses and immunocytochemistry. Type VII collagen gene expression was greatly enhanced in all cell strains studied after stimulation by transforming growth factor-beta (TGF-beta). However, no definitive correlation between the donor age and the magnitude of TGF-beta response could be made. In contrast, the basal expression of the type VII collagen gene was shown to decrease in an age-dependent manner in fibroblasts. The pro-inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) were shown to elevate type VII collagen mRNA levels in a dose-dependent manner. This response was inversely related to the donor age of the cell cultures. The attenuated response of cells from older individuals to TNF-alpha and IL-1 beta was specific for type VII collagen gene expression, because, in the same experiments, collagenase gene expression was strongly elevated by the two cytokines. Our data suggest that type VII collagen gene expression is subject to modulation by the cytokine network, which may play a role in controlling anchoring fibril assembly in normal skin and in pathologic conditions characterized by altered deposition of type VII collagen.
Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts.
The effects of acidic and basic fibroblast growth factors (FGFs) on collagen expression by keloid fibroblasts were examined in the absence and presence of heparin. Collagen biosynthesis and gene expression of type I collagen were down-regulated by the FGFs in the presence of heparin. Acidic FGF, in a concentration range of 0.4 to 50 ng/ml, had little or no effect on collagen synthesis after a 4-day incubation. However, in the presence of heparin (100 micrograms/ml) acidic FGF, in concentrations ranging from 2 to 50 ng/ml, decreased [3H]hydroxyproline synthesis by 44 to 68%, compared with untreated control cultures. Total [3H]hydroxyproline synthesis was similar between control and heparin-treated cultures. Basic FGF (2.0 to 50 ng/ml) was effective in suppressing [3H]hydroxyproline synthesis by 50 to 90% after a 4-day incubation without heparin in keloid and normal fibroblast cultures. The steady-state levels of type I collagen messenger RNA were significantly decreased by acidic FGF in the presence of heparin, as well as by basic FGF without heparin. The data suggest that the FGFs are effective in down-regulating excess collagen production by keloid fibroblasts and that this inhibitory effect is apparently associated with pretranslational events. Moreover, acidic FGF is apparently dependent on heparin, whereas basic FGF is not, for potentiation of the down-regulatory effects of the FGFs.
Type VII collagen gene expression in human umbilical tissue and cells.
BACKGROUND: Type VII collagen is a minor collagen found in anchoring fibrils. It is expressed predominantly by keratinocytes. In this study, we report the localization and spatial distribution of type VII collagen gene expression in the human umbilical cord, a fetal-derived tissue. EXPERIMENTAL DESIGN: Human umbilical cords were examined in indirect immunofluorescence studies, employing a mouse monoclonal anti-human type VII collagen antibody. Endothelial cells were cultured from the vein and grown on chamber slides for the detection of type VII collagen epitopes. In addition, cultured human umbilical vein cells were analyzed by Northern transfer analysis and by polymerase chain reaction for the expression of the corresponding gene. Fibroblast-like cells were isolated from the Wharton's jelly and were analyzed similarly for type VII collagen expression as well. RESULTS: We demonstrate that type VII collagen is expressed by human umbilical tissue and cells. Indirect immunofluorescence studies demonstrate the presence of type VII collagen epitopes in the epithelium surrounding a connective tissue region known as Wharton's jelly. In addition, there was low but detectable immunofluorescence signal associated with endothelial cells of blood vessels within the umbilical cord. In vitro, the fibroblast-like cells cultured from the Wharton's jelly showed prominent type VII collagen signal. This result was supported by the finding of high level of type VII collagen mRNA in these cells. The human endothelial cells from the vein demonstrated weak but detectable staining for type VII collagen, and the corresponding gene expression was shown by polymerase chain reaction analysis of the mRNA of the endothelial cells. CONCLUSIONS: The results show that umbilical tissue and cells, specifically those from the Wharton's jelly, are relatively enriched in type VII collagen. There is differential spatial localization of this collagen in the fetal tissue. The novel finding is that cells, other than epithelial cells such as keratinocytes, are able to express the type VII collagen gene.
Elevated expression of beta 1 and beta 2 integrins, intercellular adhesion molecule 1, and endothelial leukocyte adhesion molecule 1 in the skin of patients with systemic sclerosis of recent onset.
OBJECTIVE: To investigate the possible role of integrins and cell adhesion molecules in the pathogenesis of the mononuclear cell infiltration and fibrosis of skin that occurs in systemic sclerosis (SSc). METHODS: The presence and topographic distribution of beta 1, beta 2, and beta 4 integrins, as well as of endothelial leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1), was examined immunohistochemically in affected skin from 8 patients with rapidly progressive SSc of recent onset. The expression of the beta 1 integrin gene was also investigated by in situ hybridization with a human sequence-specific complementary DNA. RESULTS: The presence of beta 1 integrin epitopes and the corresponding messenger RNA within inflammatory cells surrounding small vessels was demonstrated in SSc skin but not in normal skin. Lymphocytes positive for beta 2 integrin were also found only in SSc skin, and they appeared in close proximity to small blood vessels and collagen bundles. Immunostaining for beta 4 integrin epitopes revealed no differences between normal and SSc skin. ELAM-1 and ICAM-1 monoclonal antibodies, which identify epitopes indicative of endothelial cell activation, stained endothelial cells in SSc skin but not normal skin. CONCLUSION: These observations suggest that the complex interactions of beta 1 and beta 2 integrins, as well as ELAM-1 and ICAM-1, may be intimately involved in the pathogenesis of SSc, perhaps by mediating the homing and targeting of pathogenetic lymphocytes to the affected tissues.
Differential expression of laminin isoforms and beta 4 integrin epitopes in the basement membrane zone of normal human skin and basal cell carcinomas.
The basement membrane zone biology of normal human skin and basal cell carcinomas was explored by indirect immunofluorescence with monoclonal antibodies recognizing five subunit polypeptides of three different laminin isoforms as well as the beta 4 integrin epitopes. The laminin antibodies were specific for A, B1, and B2 chains of classic laminin, for the M chain of merosin, or for the S chain in S-laminin. Immunostaining of normal human skin revealed a strong signal with antibodies for A, B1, and B2 chain epitopes. A weak immunosignal was detected with an anti-M chain antibody, whereas the S-chain epitopes were undetectable, even following pretreatment of sections with hyaluronidase. Thus, the laminin at the epidermal-dermal junction of normal human skin is primarily of the classic type, with some merosin molecules being present. The staining of six nodular basal cell carcinomas revealed the presence of A, B1, and B2 chain epitopes in a linear pattern, but, in contrast to normal skin, the antibody recognizing M-chain epitopes yielded a strong immunosignal, and S-chain epitopes could also be readily detected. Staining for beta 4 integrins, potential receptors for laminin, revealed a strong staining reaction in normal skin as well as in the superficial portions of the basal cell carcinoma. However, the immunofluorescence pattern in the deeper portions of the lesions was scattered and interrupted. Thus, altered composition of the basement membrane of nodular basal cell carcinomas with respect to laminin isoforms and their interactions with putative cell-surface receptors, the beta 4 integrins, may change the containment of the tumor islands, contributing to the local aggressive behavior of basal cell carcinomas.
Transforming growth factor-beta up-regulates the expression of the genes for beta 4 integrin and bullous pemphigoid antigens (BPAG1 and BPAG2) in normal and transformed human keratinocytes.
Three distinct proteins, namely, beta 4 integrins, and the 230-kDa (BPAG1) and 180-kDa (BPAG2) bullous and pemphigoid antigens, have been shown to co-localize with hemidesmosomes at the dermal-epidermal basement membrane zone. In this study, we examined the expression of the corresponding genes in cultures of normal and transformed human epidermal keratinocytes. The expression of these genes was detected by Northern and in situ hybridizations, and the expression of beta 4 integrins was also demonstrated by indirect immunofluorescence. The results indicated clearly detectable expression of all three genes in normal keratinocytes, whereas extremely low or undetectable levels of expression were noted in two transformed cell lines. Addition of TGF-beta 1 or TGF-beta 2 (10 ng/ml) up-regulated mRNA levels for all three proteins (up to 4.6 times). The increase by TGF-beta 1 was particularly striking in keratinocyte cultures incubated in the presence of low (0.15 mM) Ca++, and somewhat less pronounced in the presence of high (1.2 mM) Ca++. The increase in beta 4 integrin synthesis was also documented by enhanced immunosignal of the corresponding epitopes. These results indicate that the three hemidesmosomal genes studied here are all responsive to TGF-beta. These observations, together with previous data on the effects of TGF-beta on other components of the skin, suggest that this cytokine may play a role in the development and repair of the cutaneous basement membrane zone.
Type VII collagen gene expression by cultured human cells and in fetal skin. Abundant mRNA and protein levels in epidermal keratinocytes.
Type VII collagen, a genetically distinct member of the collagen family, is present in the cutaneous basement membrane zone as an integral component of the anchoring fibrils. We have recently isolated several cDNAs that correspond to human type VII collagen sequences. One of these cDNAs (clone K-131) was utilized to examine type VII collagen gene expression in cultures of human cells by Northern analyses, in situ hybridizations and indirect immunofluorescence. Northern hybridizations revealed the presence of an approximately 9-kb mRNA transcript, and indicated a high level of expression in epidermal keratinocytes as well as in an oral epidermoid carcinoma cell line (KB), while the expression was considerably lower in skin fibroblasts and in several virally or spontaneously transformed epithelial cell lines. In situ hybridizations of cultured keratinocytes supported the notion of a high level of gene expression. Indirect immunofluorescence of skin from a 19-wk fetus revealed type VII collagen gene expression at the dermal-epidermal basement membrane zone. These results indicate that several different cell types including epidermal keratinocytes and dermal fibroblasts express the type VII collagen gene, but epidermal keratinocytes may be the primary cell source of type VII collagen in developing human skin.
Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.
Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.
Glucose transporters of rat peripheral nerve. Differential expression of GLUT1 gene by Schwann cells and perineural cells in vivo and in vitro.
Expression of GLUTs in rat peripheral nerve was first studied at the mRNA level with Northern transfer analysis with cDNAs specific for GLUT1, GLUT2, GLUT3, and GLUT4. GLUT1 mRNA was the only GLUT mRNA detectable in rat sciatic nerve. In situ hybridization localized this mRNA to the perineurium and to some endo- and epineurial capillaries. Indirect immunofluorescence stainings demonstrated that GLUT1 protein epitopes were concentrated primarily in the perineurium and endoneurial capillaries. Also, some Schwann cells, a few epineurial capillaries, and medium-sized blood vessels showed a faintly positive immunoreaction. All cell types present in primary cultures initiated from rat sciatic nerve (perineurial cells, Schwann cells, and fibroblasts) expressed GLUT1 protein in vitro. Thus, Schwann cells, which expressed GLUT1 only occasionally at a low level in vivo, have the potential to express GLUT1 at a markedly higher level under cell culture conditions. Incubation of the cultures in 25 mM D-glucose for 7 days caused a 39% reduction in the amount of immunodetectable GLUT1 protein, and a marked (34%) decrease of GLUT1 mRNA compared with cultures incubated in 5.5 mM D-glucose. Interestingly, the reduction of [3H]-2-DG uptake in the same cultures exceeded 70%, suggesting that the reduced amount of GLUT1 protein alone did not explain the marked reduction in glucose uptake in these cultures. Immunostaining of the cell cultures suggested that perineurial cells were the main target for the glucose-induced decrease of GLUT1 protein.
Transforming growth factor-beta up-regulates type VII collagen gene expression in normal and transformed epidermal keratinocytes in culture.
Transforming growth factor-betas (TGF-betas) have been shown to enhance the expression of extracellular matrix genes, including several collagens. In this study, the effects of TGF-beta 1 and TGF-beta 2 on the expression of the gene for type VII collagen, the major component of anchoring fibrils, in human epidermal cell cultures were examined. Incubation of human epidermal keratinocytes or oral epidermoid carcinoma KB cells with TGF-beta 1 or TGF-beta 2 markedly (up to 6.3-fold) elevated the alpha 1(VII) collagen mRNA levels. This elevation was accompanied by enhanced synthesis of type VII collagen, as demonstrated by indirect immunofluorescence with a monoclonal antibody. The results indicate that TGF-beta 1 and TGF-beta 2 have similar biological activities with respect to enhanced type VII collagen gene expression.
Extracellular matrix gene expression by human endothelial and smooth muscle cells.
In this study, the expression of extracellular matrix genes by vascular cells from human iliac blood vessels was characterized on the mRNA steady-state level by slot blot and Northern transfer analyses, as well as by in situ hybridization. Endothelial cells were isolated from adult human iliac arteries and veins, as well as from umbilical veins; smooth muscle cells were isolated from adult human iliac arteries and inferior vena cava. The results show that confluent umbilical vein endothelial cells expressed the genes that encode types I, III, IV and VI collagens, as well as fibronectin and laminin. In contrast, the iliac endothelial cells expressed the genes for types IV and V collagens, fibronectin and laminin; mRNA transcripts for types I, III and VI collagens were not detectable. The smooth muscle cells from iliac arteries or inferior vena cava displayed gene expression for types I, III, IV, V and VI collagens, fibronectin and laminin. The results indicate major differences in gene expression for the various types of collagens by human iliac endothelial and smooth muscle cells. Furthermore, the fetal-derived umbilical endothelial cells displayed differential collagen gene expression from that of adult iliac endothelial cells.
Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-beta 1 mRNA.
Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.
Transforming growth factor-beta improves healing of radiation-impaired wounds.
Exogenously applied TGF-beta 1 has been shown to increase wound strength in incisional wounds early in the healing process. An impaired wound healing model was first established in guinea pigs by isolating flaps of skin and irradiating the flaps to 15 Gray in one fraction using a 4-MeV linear accelerator. Incisions made 2 d after irradiation were excised 7 d later, and showed decreased linear wound bursting strength (WBS) as compared to non-irradiated control wounds on the contralateral side of each animal (p = 0.001). The effect of TGF-beta on healing of radiation-impaired wounds was studied using this model. Skin on both left and right sides of guinea pigs was irradiated as above. A linear incision was made in each side. Collagen with either 1, 5, or 20 micrograms of TGF-beta was applied to one side prior to closure with staples, whereas the contralateral side received saline in collagen. Wounds given either 1 or 5 micrograms of TGF-beta were found to be stronger than controls at 7 d (p less than 0.05), whereas those receiving the higher 20-micrograms dose were weaker than controls (p less than 0.05). Thus, TGF-beta in lower doses improved healing at 7 d but very large amounts of the growth factor actually impaired healing. In situ hybridization done on wound samples showed increased type I collagen gene expression by fibroblasts in wounds treated with 1 micrograms TGF-beta over control wounds. These results indicate that TGF-beta improved wound healing as demonstrated by increased WBS. This improvement is accompanied by an up-regulation of collagen gene expression by resident fibroblasts.
Elevated expression of the genes for transforming growth factor-beta 1 and type VI collagen in diffuse fasciitis associated with the eosinophilia-myalgia syndrome.
Full-thickness skin biopsies obtained from four patients with rapidly progressive diffuse fasciitis associated with the Eosinophilia-Myalgia syndrome (EMS) were examined for the expression of transforming growth factor-beta 1 (TGF-beta 1), type VI collagen, and fibronectin genes employing immunohistochemistry and in situ hybridizations. The immunohistochemical studies demonstrated increased deposition of TGF-beta, type VI collagen, and fibronectin epitopes in the extracellular matrix of the fascia in comparison to the adjacent dermis in the same specimens. Increased levels of type VI collagen mRNA, as evidenced by positive in situ hybridization signals with an alpha 2(VI) collagen cDNA, were also found in the fascia in comparison with the dermis. In situ hybridizations of affected fascia with a human sequence-specific TGF-beta 1 cDNA demonstrated numerous fibroblasts displaying positive hybridization signals indicative of high levels of transcripts for this cytokine. In contrast, no hybridization signal for TGF-beta 1 was detected in fibroblasts in the adjacent dermis. These findings suggest that TGF-beta 1 may play an important role in the development of the connective tissue alterations present in EMS-associated diffuse fasciitis.