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Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 397 records · Page 22Linked to original sources
PubMed1989

Molecular mechanisms of cutaneous aging. Age-associated connective tissue alterations in the dermis.

Clinically detectable, age-associated cutaneous changes result from two independent processes: chronologic aging and actinic irradiation. Several lines of evidence suggest that these two processes have different biologic, biochemical, and molecular mechanisms. This review summarizes the current understanding of age-associated alterations in the biochemistry and molecular biology of the extracellular matrix.

Aged↗
PubMed1988

Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide.

Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approximately 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutis laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

Base Sequence↗
PubMed1988

Modulation of collagen metabolism by glucocorticoids. Receptor-mediated effects of dexamethasone on collagen biosynthesis in chick embryo fibroblasts and chondrocytes.

The steroid modulation of collagen metabolism was studied by injecting chick embryos with dexamethasone in vivo, and collagen synthesis was subsequently assayed by pulse-labeling the tissue with [14C]proline in vitro. The synthesis of [14C]hydroxyproline in tendons and sterna from chick embryos treated with dexamethasone was markedly reduced as compared with untreated controls. The inhibition of [3H]hydroxyproline synthesis was accompanied by a similar reduction in type I and II procollagen mRNA levels, as detected by Northern blot and dot blot hybridizations with chick pro alpha 1(I), pro alpha 2(I) and pro alpha 1(II) sequence specific cDNAs. The reduction in type II procollagen mRNA level was shown to be dose dependent. Control experiments indicated that the post-translational hydroxylation of prolyl residues was only slightly decreased in dexamethasone treated animals, and that the specific activity of the intracellular free proline pool and the intracellular degradation of collagen were unchanged. To address the mechanisms of the inhibition of collagen biosynthesis, specific binding of dexamethasone to glucocorticoid receptors in chick embryo tendon and cartilage cells was studied in a whole cell assay using [3H]dexamethasone as the ligand. Matrix-free tendon and cartilage cells had approximately 19,000 and 15,000 receptor sites per cell, respectively, and the binding affinities (Kd) for dexamethasone in tendon and cartilage cells were 2.9 x 10(-9) and 2.3 x 10(-9) M. Comparable values were obtained using a cytosol binding assay. The nuclear binding of dexamethasone in tendon and cartilage cells were similar. The results suggest that the dexamethasone-induced inhibition of collagen production is primarily due to decreased levels of functional procollagen mRNA, possibly resulting from receptor-mediated inhibition of the gene expression on the transcriptional level.

Animals↗
PubMed1988

Werner's syndrome. Evidence for preferential regional expression of a generalized mesenchymal cell defect.

We describe a patient with Werner's syndrome from whom skin biopsy specimens were sampled for histology and electron microscopy and fibroblasts were cultured. Tissue sampled from five sites that varied in clinical presentation revealed striking changes in the dermoepidermal junction, elastic fibers of the papillary and reticular dermis, and adipose tissue of the hypodermis. The density and organization of the collagenous connective tissue was altered variably depending on the biopsy site. Changes noted in the epidermis were indicative of tissue regeneration and repair. Cells derived from acral areas grew poorly and could not be passed. Collagen synthesis in these cells was enhanced approximately 50%, and collagenase expression was decreased to a similar degree. Cells derived from the skin of the trunk could be passed but had an abbreviated in vitro life span. Collagen synthesis in these cells was unaltered. Serum from the patient with Werner's syndrome or from his obligate heterozygote offspring stimulated collagen synthesis in low-passage normal human skin fibroblast target cells. Sequential passage of these normal cells resulted in a blunting of the stimulatory effect. These observations suggest that a stimulator of collagen synthesis exists in the serum of patients with Werner's syndrome and that as cells (either normal or Werner's syndrome) "age" in vitro they may become hyporesponsive to this as yet undefined stimulatory factor in serum.

Collagen↗
PubMed1988

Mechanism of tissue fusion in argon laser-welded vein-artery anastomoses.

The mechanism of laser vascular tissue welding remains unknown. This study compared the acute tissue response and long-term healing of sutured and laser-welded anastomoses of vein segments used to bypass ligated canine femoral arteries. For each procedure, one anastomosis was formed using running 6-0 polypropylene suture (control), and the other anastomosis was formed using argon laser tissue welding (experimental). The vein grafts were harvested at 4 (n = 2), 8 (n = 1), 12 (n = 1), and 16 (n = 2) weeks, and selected samples were evaluated by histologic examination, electron microscopy, tensile strength testing, and by measuring the formation of [3H]hydroxyproline as an index of collagen synthesis. Examination of successful laser fusions immediately after they were formed showed bonding of collagen to collagen and elastin to collagen. Follow-up evaluations showed that the precision of tissue apposition affected the rate of healing and tensile strength. Laser-welded anastomoses demonstrated a progression of healing similar to sutured repairs, with remodeling of fibrous tissue and collagen being the primary component of weld integrity. This study demonstrates that sutured and argon laser-welded vein-artery anastomoses heal comparably up to 16 weeks postoperatively, and that laser welding is a satisfactory alternative to sutured anastomoses.

Animals↗
PubMed1988

Cellular effects of the pulsed tunable dye laser at 577 nanometers on human endothelial cells, fibroblasts, and erythrocytes: an in vitro study.

The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 microseconds is rapidly becoming the treatment of choice for removal of portwine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells--erythrocytes, endothelial cells, and fibroblasts. Human endothelial cells and fibroblasts in microwell plates were irradiated at various energy densities with the laser, after which several aspects of cellular biology were determined, including 1) viability of cells by trypan blue exclusion test; 2) cell proliferation by [3H]thymidine incorporation; and 3) rate of protein synthesis using [3H]leucine incorporation as a marker. In endothelial cell cultures, both [3H]thymidine and [3H]leucine incorporations were inhibited at energy levels of 5-12 J/cm2 (P less than 0.01). In fibroblast cultures, cell proliferation was similarly inhibited, while supratherapeutic energy density (greater than or equal to 12 J/cm2) was required for inhibition of protein synthesis. The laser energy in the range of 5-8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis, as determined by release of free hemoglobin into culture medium. Addition of erythrocytes into a coculture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells were present alone. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm.

Cell Division↗
PubMed1988

Ultrastructural characteristics of keloid fibroblasts.

Myofibroblasts abound in granulation tissue, where they play an important role in the healing process. We evaluated the characteristics of fibroblasts in ten patients with keloids, a disorder of skin wound repair. Keloid lesions uniformly exhibited numerous fibroblasts with prominent Golgi complexes and abundant rough endoplasmic reticulum. Although rare fibroblasts exhibited a convoluted nucleus and tangled microfilaments, none could be characterized as myofibroblast, as defined by cytoplasmic myofilaments and cell to cell junctions. We conclude that fibroblast density and activity are increased in keloids, but true myofibroblasts are not a component of these lesions.

Adult↗
PubMed1988

Fibronectin gene expression by epithelial tumor cells in basal cell carcinoma: an immunocytochemical and in situ hybridization study.

Previous observations have demonstrated that fibronectin is deposited in high abundance in basal cell carcinoma stroma. In this study, the nature of fibronectin and the site of its synthesis were explored in 10 basal cell carcinomas of the nodulo-ulcerative type by immunocytochemistry and in situ hybridization. First, simultaneous localization of epithelial tumor cell islands and fibronectin epitopes was carried out by double immunofluorescence staining with monoclonal anti-cytokeratin antibodies and polyclonal fibronectin antibodies, the latter recognizing both the cellular and plasma types of the protein. Large amounts of fibronectin were deposited in the basal cell carcinoma stroma, with the highest concentration present in the immediate proximity of the epithelial cell islands. Immunofluorescence with a monoclonal anti-fibronectin antibody, which is directed against the ED-domain of cellular fibronectin and does not recognize the plasma type of fibronectin, revealed essentially the same staining pattern as that obtained with the polyclonal anti-fibronectin antibody. This observation suggested that fibronectin in BCC was predominantly of the cellular type. Second, in situ hybridizations, utilizing a human fibronectin specific cDNA, demonstrated that the highest concentration of fibronectin mRNA was found in the most peripheral cell layer of the epithelial tumor islands. The presence of fibronectin mRNAs in the tumor cells of the central regions of the islands, as well as within occasional stromal cells, was also noted. Thus, two lines of evidence suggest that the epithelial tumor cells are predominantly responsible for the synthesis and deposition of fibronectin in basal cell carcinoma. The presence of fibronectin may explain the characteristic biologic behavior of basal cell carcinomas, including low degree of metastatic potential and local destructive nature of the tumors.

Autoradiography↗
PubMed1988

Cloning of full-length elastin cDNAs from a human skin fibroblast recombinant cDNA library: further elucidation of alternative splicing utilizing exon-specific oligonucleotides.

A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.

Amino Acid Sequence↗
PubMed1988

Selectively enhanced procollagen gene expression in sclerosing (morphea-like) basal cell carcinoma as reflected by elevated pro alpha 1(I) and pro alpha 1(III) procollagen messenger RNA steady-state levels.

Sclerosing or morphea-like variant of basal cell carcinoma (BCC) is characterized by an extensive connective tissue stroma, and histopathology has suggested that the extracellular matrix is largely composed of collagen. In addition, fibronectin deposition has been proposed to modulate tumor growth in BCC. In this study, we examined the expression of genes coding for type I, III, and IV procollagens, as well as for fibronectin, in tissue from 10 patients with sclerosing BCC. For comparison, tissues from 5 patients with nodular BCC and 4 controls were examined. Total RNA was isolated by CsCl density gradient centrifugation, and messenger RNA (mRNA) steady-state levels were determined by slot-blot hybridizations with human sequence specific complementary DNAs (cDNAs). The abundance of type I procollagen mRNA in sclerosing BCC tissue was increased to 233.6 +/- 36.7% of the controls (mean +/- SEM). The corresponding value for type III procollagen mRNA in sclerosing BCC was 281.8 +/- 54.8% of the controls. Consequently, the steady-state ratio of type I/III procollagen mRNAs in sclerosing BCCs (5.0 +/- 1.2; mean +/- SD) was within the control range. Thus, there is a coordinate increase in type I and type III procollagen mRNA levels in sclerosing BCC. In contrast, the values for type I and type III procollagen mRNAs in nodular BCC were not different from the controls. In addition, type IV procollagen and fibronectin mRNA levels were not different from the controls either in sclerosing or nodular BCCs, attesting to the selectivity of the increase in type I and III procollagen mRNA levels in sclerosing BCC. These observations may relate to the excessive deposition of the extracellular matrix stroma surrounding the tumor cells in sclerosing BCC.

Basal Cell Carcinoma↗
PubMed1988

Expression of basement membrane zone genes coding for type IV procollagen and laminin by human skin fibroblasts in vitro: elevated alpha 1 (IV) collagen mRNA levels in lipoid proteinosis.

Basement membrane zone gene expression by fibroblast cultures established from adult human skin was examined by molecular hybridizations with human sequence-specific cDNAs corresponding to pro-alpha 1 (IV) chain of type IV collagen and B2 chain of laminin. Northern transfer analysis of poly(A)+RNA isolated from fibroblast cultures clearly revealed the presence of specific mRNA transcripts, indicating the expression of the genes coding for pro-alpha 1 (IV) and laminin B2 polypeptides. Quantitative estimates of the relative levels of these mRNAs coding for basement membrane zone components indicated that they are of relatively low abundance in control fibroblasts, as compared with mRNAs coding for fibronectin or pro-alpha 1 (I) chain of type I procollagen. In fibroblast cultures established from the lesional skin of a 32-year-old patient with lipoid proteinosis, the levels of mRNA coding for pro-alpha 1 (IV) polypeptides were increased approximately 4.5-fold, as compared with age- and passage-matched control cultures. This increase was selective in that the levels of fibronectin, pro-alpha 1 (I), and beta-actin mRNAs were unaltered in the same cultures. The increase in pro-alpha 1 (IV) mRNA level was also uncoordinate with the expression of the laminin B2 chain gene, which was unaltered in lipoid proteinosis. The selective increase in pro-alpha 1 (IV) mRNA may have relevance to the accumulation of this basement membrane component in the skin in lipoid proteinosis.

Adult↗
PubMed1988

The flashlamp-pumped 577-nm pulsed tunable dye laser: clinical efficacy and in vitro studies.

The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 microseconds is rapidly becoming the treatment of choice for removal of port-wine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells in vitro: erythrocytes, endothelial cells, and fibroblasts. Clinical studies were also performed, addressing the efficacy of this laser in the treatment of port-wine stains of the head and neck, trunk, and extremities. In endothelial cell cultures, both [3H]thymidine (measuring cell proliferation) and [3H]leucine (measuring protein synthesis) incorporations were inhibited at energy levels of 5-12 J/cm2 (p less than 0.01). The laser energy in the range of 5-8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis. Addition of erythrocytes into a co-culture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells alone were present. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm. Clinical trials on 28 patients with port-wine stains of the face and neck using this laser demonstrated a 75% response rate with greater than 50% lightening of the lesions. Of 9 port-wine stain lesions on the trunk and extremities (on a total of 6 patients), 8 (89%) demonstrated significant (greater than 50%) fading of their lesions. Complications such as scarring or epidermal texture changes were minimal.

Adult↗
PubMed1988

Cellular differentiation and expression of matrix genes in type 1 neurofibromatosis.

The cellular heterogeneity of cutaneous tumors from nine patients with type 1 (von Recklinghausen's) neurofibromatosis was studied using several antigenic markers with special reference to focal heterotopic differentiation and interindividual variation. Furthermore, cells which actively express the genes for type I and III collagens and fibronectin, the major components of the abundant extracellular matrix of neurofibromas, were localized using in situ hybridizations. In eight of nine cases, the S-100 protein positive cells, i.e. Schwann-like cells, composed 60 to 80% of the total cell population. However, in one case, only about 40% of the cells were S-100 protein positive. The latter tumor was studied with respect to perineurial cell differentiation and stained with a mixture of two antibodies, directed against the S-100 protein and type IV collagen. In Schwann cells, the staining reaction for S-100 protein was observed in the nuclear region, whereas the staining reaction for type IV collagen was located peripherally, corresponding to the basement membrane zone covering the cells. The stromal cells which showed only the peripheral staining profile were considered to be neoplastic perineurial cells. Distinct structures with epithelial, endothelial, or smooth muscle cell differentiation were present within the benign tumors, as detected by immunostaining for cytokeratin, epithelial membrane antigen, factor VIII-related antigen and desmin, respectively. In situ hybridizations revealed a clearly detectable expression of type I procollagen genes in less than 10% and type III procollagen gene in less than 5% of the total cell population. Active synthesis of fibronectin was limited to the vascular walls, when examined by in situ hybridization, and antibodies to cellular fibronectin localized to the same areas. However, antibodies to plasma fibronectin produced a uniform staining reaction throughout the tumors suggesting that most of the fibronectin in neurofibromas is plasma-derived. The latter observation suggests that neurofibroma cells are freely accessible to various plasma proteins, including growth factors, which may influence the growth characteristics of these lesions.

Antibodies, Monoclonal↗
PubMed1988

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.

Aging↗
PubMed1987

Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action.

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.

Collagen↗
PubMed1987

Scleredema and paraproteinemia. Enhanced collagen production and elevated type I procollagen messenger RNA level in fibroblasts grown from cultures from the fibrotic skin of a patient.

An edematous rash developed on the abdominal skin of a 76-year-old woman who had had diabetes mellitus for ten years. Some months later, the affected skin became thickened and indurated. Histopathologic examination revealed marked dermal fibrosis with excessive deposition of collagen. The patient also had IgA (k-type) paraproteinemia. Fibroblast cultures from the affected and unaffected skin were studied for collagen metabolism. Procollagen synthesis was elevated about sixfold on fibroblasts derived from the affected skin. A similar increase was detected in messenger RNA (mRNA) levels using a complementary DNA clone specific for human pro alpha 1(l) collagen mRNA. The elevated mRNA level could be the result of increased transcriptional activity of collagen genes or decreased degradation of collagen mRNAs. Our findings suggest that increased collagen deposition may account for the marked dermal fibrosis that we observed in this patient.

Aged↗
PubMed1987

Procollagen gene expression by scleroderma fibroblasts in culture. Inhibition of collagen production and reduction of pro alpha 1(I) and pro alpha 1(III) collagen messenger RNA steady-state levels by retinoids.

Recent studies have demonstrated that retinoids (synthetic vitamin A analogs) can modulate connective tissue metabolism in human skin fibroblast cultures. In this study, we examined the effects of 3 retinoids, all-trans-retinoic acid (RA), 13-cis-RA, and an aromatic retinoid, RO-10-9359, on collagen gene expression in scleroderma fibroblast cultures and matched control fibroblast cultures. The results indicated that all-trans-RA and 13-cis-RA significantly reduced procollagen production both in control and scleroderma fibroblast cultures in a dose-dependent manner. The reduction in procollagen production was paralleled by a similar decrease in steady-state levels of type I and type III procollagen messenger RNAs, which suggests that there is coordinate inhibition on the transcriptional level. In contrast, RO-10-9359 elicited only limited effects on collagen production, and such effects were variable. The results suggest that further development of retinoids might provide an effective means to counteract tissue deposition of collagen in scleroderma and other fibrotic diseases.

Adult↗