RESUMO
Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.
Assuntos
Colágeno/antagonistas & inibidores , Regulação da Expressão Gênica , Pró-Colágeno/antagonistas & inibidores , Pele/citologia , Tretinoína/farmacologia , Células Cultivadas , Colágeno/biossíntese , Endopeptidases/biossíntese , Fibroblastos/metabolismo , Humanos , Queloide/metabolismo , Manose/metabolismo , Colagenase Microbiana/antagonistas & inibidores , Neprilisina , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , RNA Mensageiro/metabolismoRESUMO
The photophysical properties and solution thermodynamics of water soluble trivalent americium (Am(III)) complexes formed with multidentate chromophore-bearing ligands, 3,4,3-LI(1,2-HOPO), Enterobactin, and 5-LIO(Me-3,2-HOPO), were investigated. The three chelators were shown to act as antenna chromophores for Am(III), generating sensitized luminescence emission from the metal upon complexation, with very short lifetimes ranging from 33 to 42 ns and low luminescence quantum yields (10(-3) to 10(-2)%), characteristic of Near Infra-Red emitters in similar systems. The specific emission peak of Am(III) assigned to the (5)D1 â (7)F1 f-f transition was exploited to characterize the high proton-independent stability of the complex formed with the most efficient sensitizer 3,4,3-LI(1,2-HOPO), with a log ß110 = 20.4 ± 0.2 value. In addition, the optical and solution thermodynamic features of these Am(III) complexes, combined with density functional theory calculations, were used to probe the influence of electronic structure on coordination properties across the f-element series and to gain insight into ligand field effects.
RESUMO
Dermal fibrosis, characterized by collagen accumulation, is the hallmark of several cutaneous diseases. To examine the mechanisms of collagen deposition in fibrotic skin diseases, fibroblast cultures were established from the skin of patients with progressive systemic sclerosis, morphea, scleredema, familial cutaneous collagenoma, connective tissue nevi of the collagen type, or keloids; these patients served as prototypes of fibrotic skin diseases with varying clinical features and potentially different etiologic factors. Collagen production was assayed by the synthesis of [3H]hydroxyproline, and types I and III procollagen messenger RNA (mRNA) levels were determined by dot blot hybridizations using human type I and type III procollagen-specific cDNA probes. The collagen production in fibroblast cultures from the fibrotic diseases was increased up to 6-fold over the controls, and a relatively good correlation between the collagen production and type I collagen mRNA levels was noted. The type I/III procollagen mRNA ratio in control fibroblast cultures was 5.9 +/- 1.6 (mean +/- SD). The corresponding ratio in keloid cell culture was markedly increased, while slightly decreased values were noted in the case of morphea and familial cutaneous collagenoma; the values in other cultures were within the normal range. The results suggest that procollagen production in fibroblast cultures derived from fibrotic skin diseases reflects elevated levels of the corresponding procollagen mRNA. The increased mRNA abundance, suggesting pretranslational control, may result from enhanced transcriptional activity of the corresponding gene or alternatively reflects increased stability of the mRNA molecule.
Assuntos
Colágeno/genética , Regulação da Expressão Gênica , Biossíntese de Proteínas , Dermatopatias/genética , Fibrose , Humanos , Hibridização de Ácido Nucleico , Pró-Colágeno/biossíntese , Dermatopatias/patologiaRESUMO
Keloids are histologically characterized by an abundance of the extracellular matrix of connective tissue. In the present study, we examined the connective tissue composition of keloids, and analyzed the details of collagen metabolism utilizing fibroblast cultures established from keloid tissue. Quantitative connective tissue analyses indicated that collagen was the predominant extracellular matrix component in keloids. The ratio of genetically distinct collagens type I/III was significantly increased, as compared to normal human skin. Collagen biosynthesis was measured in fibroblast cultures by the formation of radioactive hydroxyproline: 5 of 9 keloid cell cultures studied demonstrated increased procollagen production in comparison to age-, sex-, and passage-matched control skin fibroblast lines, while the remaining 4 cell lines were within the control range. Keloid fibroblast cultures which were high collagen producers also demonstrated elevated prolyl hydroxylase activity. The mechanisms of increased procollagen production in fibroblast cultures were first examined by assaying the abundance of type I procollagen-specific mRNA utilizing dot blot hybridizations with a pro alpha 2(I)-chain-specific cDNA. The type I procollagen mRNA levels were significantly increased in 4 keloid fibroblast lines, and a good correlation between the mRNA levels and the rate of procollagen production in the same cultures was noted. These observations suggest regulation of the collagen gene expression on the transcriptional level. The catabolic pathway of collagen metabolism in fibroblast cultures was examined by determining the degradation of newly synthesized procollagen polypeptides through assay of radioactive hydroxyproline in small-molecular-weight peptide fragments. In 3 keloid cell cultures, the degradation of newly synthesized collagen polypeptides was below the range of normal controls. These findings suggest that a reduced degradation of newly synthesized polypeptides might contribute to the accumulation of procollagen in some keloid fibroblast cultures. The results of this study suggest two possible mechanisms for deposition of collagen in keloid lesions in vivo: first, the growth of the lesions may result from a localized loss of control of the extracellular matrix production by fibroblasts; secondly, reduced degradation of the newly synthesized procollagen polypeptides may contribute to collagen deposition in some keloids.
Assuntos
Colágeno/metabolismo , Tecido Conjuntivo/metabolismo , Queloide/metabolismo , Pele/patologia , Adulto , Células Cultivadas , Colágeno/biossíntese , Colágeno/genética , Feminino , Fibroblastos/metabolismo , Humanos , Pessoa de Meia-Idade , Pró-Colágeno/biossíntese , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , RNA Mensageiro/metabolismoRESUMO
We investigated the histologic and biochemical effects of carbon dioxide and neodymium (Nd)-YAG laser welding on the healing of venotomies. Ten canine femoral venotomies 2 cm in length were approximated and welded with 10 600-nm wavelength, 1-W power over 20 to 25 s for CO2 laser, and 1060-nm wavelength, 1-W power over 30 to 40 s for Nd-YAG laser. On removal at one to three weeks, all veins (4/4 welded by CO2 and 6/6 by Nd-YAG) were patent without hematomas. Histologic and biochemical analyses of the venous tissues demonstrated active healing at the venotomy sites. We conclude that the CO2 and Nd-YAG lasers can be used successfully to weld venotomies and may provide an alternative to conventional suture techniques for repair of vascular lesions.
Assuntos
Terapia a Laser , Veias/cirurgia , Animais , Dióxido de Carbono , Cães , Estudos de Avaliação como Assunto , Humanos , Veias/anatomia & histologia , CicatrizaçãoRESUMO
We compared the histologic features, tensile strength, and collagen synthesis of laser-welded and sutured arteriotomies. Four bilateral canine femoral or carotid arteries, 2 cm long, were studied at one through four weeks postoperatively, with one vessel (control) closed with interrupted 6-0 polypropylene sutures and the contralateral vessel (experimental) welded with an argon laser (0.5 W [1417 J/cm2], four-minute exposure per 1-cm length of incision). Histologic examination revealed that laser-welded arteriotomies had less inflammatory reaction, more normal collagen and elastin reorientation, and similar endothelial continuity when compared with the control, sutured wounds. The tensile strength of the one- and two-week laser-welded specimens was less than that of sutured wounds and became approximately equal to sutured repairs at three and four weeks. There were no significant differences in the rate of collagen synthesis. There was no evidence of abnormal healing in the laser-welded specimens, suggesting that argon laser welding may be an alternative to suture repair of arteriotomies.
Assuntos
Artérias/cirurgia , Terapia a Laser , Técnicas de Sutura , Animais , Artérias Carótidas/cirurgia , Colágeno/biossíntese , Cães , Artéria Femoral/cirurgia , Resistência à Tração , CicatrizaçãoRESUMO
Recent observations have suggested that retinoids might affect the metabolism of the extracellular matrix of connective tissues. In this study, we examined the effects of tretinoin (all-trans-retinoic acid) and isotretinoin (13-cis-retinoic acid) on the production of procollagen in keloid fibroblast cultures that were characterized by enhanced procollagen synthesis in vitro. The activities of three enzymes relevant to connective tissue metabolism, prolyl hydroxylase, collagenase, and an elastaselike neutral protease were also determined. The results demonstrated that collagen production was markedly reduced in cultures treated with either one of the retinoids. The activity of prolyl hydroxylase, a key enzyme in the intracellular biosynthesis of collagen, was not affected, while the production of collagenase was markedly reduced by the retinoids. In contrast, the activity of an elastaselike neutral protease in the cell culture medium was markedly enhanced by both retinoids. The results, therefore, indicate a differential modulation of connective tissue metabolism by retinoids in keloid cell cultures.
Assuntos
Tecido Conjuntivo/metabolismo , Queloide/metabolismo , Retinoides/farmacologia , Células Cultivadas , Colágeno/biossíntese , Células do Tecido Conjuntivo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Humanos , Pró-Colágeno/biossíntese , Fatores de TempoRESUMO
Previous studies have shown the carbon dioxide (CO2) laser to be effective in the treatment of actinic cheilitis. After CO2 laser abrasion, normal skin and marked cosmetic improvement of the lip were noted. In our study, twenty-three patients were treated with CO2 laser abrasions for cosmetic improvement of facial lines and actinic changes. Pre- and postoperative histopathologic examinations were made on two patients. Preoperative examination of specimens from actinically damaged skin showed atypical keratinocytes in the basal layer of the epidermis, with overlying dense compact orthokeratosis and parakeratosis. Abundant solar elastosis was seen in the papillary dermis. Postoperative histologic specimens showed a normal-appearing epidermis with fibrosis in the papillary dermis and minimal solar elastosis (about four weeks after laser treatment). At present, various modalities are available for the regeneration of the aged skin, including chemical peels and dermabrasion. Significantly fewer complications were noted with CO2 laser abrasion than with these methods. Thus, CO2 laser abrasion can be useful in the cosmetic and medical treatment of the aged skin. Marked clinical and histologic improvement has been demonstrated.
Assuntos
Queilite/cirurgia , Face/cirurgia , Terapia a Laser , Raios Ultravioleta/efeitos adversos , Adulto , Idoso , Biópsia , Queilite/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Hands are a primary target for actinic damage and premature aging. Until recently, very little could be done to successfully erase the various signs of photoaging. This article presents a simple technique, combining fat transfer and laser resurfacing, to safely and effectively rejuvenate hands and erase the signs of aging.
Assuntos
Tecido Adiposo/transplante , Envelhecimento/patologia , Dermatoses da Mão/cirurgia , Terapia a Laser , Atrofia , Dermatite Seborreica/cirurgia , Dermatoses da Mão/patologia , Humanos , Ceratose/cirurgia , Lentigo/cirurgiaRESUMO
Eight patients underwent carbon dioxide (CO2) laser blepharoplasty. The temperature of the upper eyelid conjunctiva was monitored and recorded during laser surgery. A minimal increase in temperature was noted. This study confirms the absence of thermal injury at a distal site from the laser application and the safety of the laser procedure.
Assuntos
Pálpebras/cirurgia , Terapia a Laser , Cirurgia Plástica , Túnica Conjuntiva/fisiologia , Humanos , Período Intraoperatório , Terapia a Laser/efeitos adversos , TemperaturaRESUMO
Cultured human lymphocytes were subjected to irradiation with a gallium-arsenide laser at energy fluence varying from 2.17 to 651 mJ/cm2, and the cell proliferation was assessed by [3H]thymidine incorporation. Both mitogenic proliferation in response to phytohemagglutinin and spontaneous cell proliferation were markedly inhibited by the laser irradiation at energy fluence as low as 10.85 mJ/cm2. Similarly, the functional response of cells to antigen stimulation in a one-way mixed-lymphocyte reaction was also diminished as a result of laser irradiation. The results indicate that laser irradiation at low energy can interfere with immune system in vitro, and similar modulation could potentially occur in human subjects exposed to laser irradiation in vivo.
Assuntos
Imunidade Celular/efeitos da radiação , Lasers , Relação Dose-Resposta à Radiação , Humanos , Técnicas In Vitro , Lasers/efeitos adversos , Ativação Linfocitária/efeitos da radiação , Teste de Cultura Mista de LinfócitosRESUMO
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.
Assuntos
Queloide/patologia , Adulto , Retículo Endoplasmático/ultraestrutura , Feminino , Fibroblastos/ultraestrutura , Complexo de Golgi/ultraestrutura , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Aortae from 17-day old chick embryos were subjected to irradiation with a Nd:YAG laser at energy densities varying from 1.2 - 4.7 X 10(3) J/cm2. The aortae were pulse-labeled in vitro with [3H]proline or [14C]valine, and the synthesis of collagenous polypeptides and soluble elastin was examined by SDS-polyacrylamide gel electrophoresis, followed by fluorography and quantitative scanning densitometry. Irradiation of the aortae with Nd:YAG laser resulted in inhibition of the synthesis of the extracellular matrix proteins. The production of collagen was inhibited to a considerably larger degree than the production of elastin. Thus, the biosynthetic pathway for collagen production appears to be more susceptible to laser inhibition than the corresponding pathway for elastin production. These observations may have relevance to laser angioplasty which has been proposed to be applicable for removal of atherosclerotic plaques in human vessels. Specifically, the results suggest that inhibition of the extracellular matrix production may result in weakening of the vessel wall with subsequent aneurysm formation and rupture.
Assuntos
Aorta/efeitos da radiação , Colágeno/biossíntese , Elastina/biossíntese , Lasers , Animais , Aorta/embriologia , Aorta/metabolismo , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Fibronectinas/biossíntese , Peso Molecular , Prolina/metabolismo , Valina/metabolismoRESUMO
The concept of tissue fusion by laser has been recently established. In this study, we have examined the skin welding by laser and tested the efficacy of four different lasers for this application. The results attest to the feasibility of the procedure and suggest that laser welding may represent an alternative for closure of cutaneous wounds.
Assuntos
Terapia a Laser , Pele/lesões , Animais , Procedimentos Cirúrgicos Dermatológicos , Camundongos , Camundongos Pelados , Técnicas de Sutura , Resistência à Tração , CicatrizaçãoRESUMO
Recent evidence suggests that low-energy lasers, such as an He-Ne laser, might stimulate wound healing. In this review we are summarizing our recent observations indicating that low-energy lasers enhance collagen gene expression both in skin fibroblast cultures in vitro, as well as in animal models of wound healing in vivo.
Assuntos
Lasers , Cicatrização/efeitos da radiação , Animais , Divisão Celular/efeitos da radiação , Células Cultivadas , Colágeno/biossíntese , Fibroblastos/efeitos da radiação , Humanos , Camundongos , Camundongos Pelados , Biossíntese de Proteínas , Resistência à Tração/efeitos da radiaçãoRESUMO
Regulation of collagen gene expression was studied in fibroblast cultures established from patients with keloids, fibrotic lesions of the skin. In selected keloid fibroblast cultures, an overproduction of type I procollagen was observed. This increase was accompanied by a parallel increase in type I procollagen-specific mRNA levels, as detected by dot-blot and RNA transfer hybridizations, without concomitant change in type I procollagen gene copy number. At the same time, type III procollagen mRNA levels were unaltered, resulting in markedly elevated type I/III procollagen mRNA ratios. Thus, keloid fibroblasts offer a unique model to study the independent regulation of the gene expression of two genetically distinct procollagens, type I and type III.
Assuntos
Queloide/metabolismo , Pró-Colágeno/genética , Células Cultivadas , Fibroblastos , Regulação da Expressão Gênica , Genes , Humanos , Queloide/genética , Pró-Colágeno/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Previous studies have indicated that laser can selectively affect the biological functions of cells. In the present study, the role of a thermal component in laser-induced alterations in the biology of human skin fibroblasts was examined. Cells were cultured on 96-well tissue culture plates, subjected to treatment with the Nd:YAG laser (wavelength 1,064 nm), and the temperature of the medium was monitored by a microprobe connected to a telethermometer . For comparison, parallel cultures were heated to the same temperatures by tungsten-halogen lamp. The cell cultures were analyzed for collagen synthesis by incubating the cultures with [3H]proline, and the collagen production was assayed by the synthesis of nondialyzable [3H]hydroxyproline. The rate of DNA replication was also determined by measuring the uptake of [3H]thymidine. A marked decrease of collagen production and thymidine incorporation was noted in the cultures subjected to Nd:YAG laser. No such decreases were noted in cultures heated to the corresponding temperatures by tungsten-halogen lamp. The results thus indicate that the biochemical alteration caused by the Nd:YAG laser in human fibroblast functions cannot be explained on the basis of thermal effects.
Assuntos
Fibroblastos/fisiologia , Lasers , Pele/citologia , Colágeno/biossíntese , Técnicas de Cultura , DNA/biossíntese , Fibroblastos/metabolismo , Halogênios , Temperatura Alta , Humanos , Hidroxiprolina/biossíntese , Neodímio , TungstênioRESUMO
Clinical observations have suggested that low-energy lasers might stimulate wound healing. To understand the mechanism of the biostimulation, we previously examined the effects of low-energy lasers on collagen production by human skin fibroblasts and reported an increase of collagen synthesis in vitro. To examine the effects of low-energy lasers in vivo, hairless mice were experimentally wounded, sutured, and subjected to laser irradiation by a helium-neon laser with a power output of 1.56 mW and an energy fluence of 1.22 Joules/cm2. Experimental wounds were subjected to laser treatment every other day for 2 months; control wounds remained untreated. Specimens from the wounds were then examined for histological findings, tensile strength, and total collagen content. Results demonstrated a considerable improvement in the tensile strength of the laser-irradiated wounds at 1 and 2 weeks. Furthermore, the total collagen content was significantly increased at 2 months when compared with control wounds. These results suggest a beneficial effect of the helium-neon laser on wound healing in vivo.
Assuntos
Terapia a Laser , Cicatrização/efeitos da radiação , Ferimentos e Lesões/radioterapia , Animais , Colágeno/metabolismo , Hélio , Hidroxiprolina/metabolismo , Camundongos , Camundongos Pelados , Modelos Biológicos , Neônio , Resistência à Tração/efeitos da radiação , Ferimentos e Lesões/metabolismoRESUMO
This study compared the histology and tensile strength of Nd:YAG laser welded and sutured small bowel enterotomies in Sprague-Dawley rats. Enterotomies (0.5 cm long) were either welded with the Nd:YAG laser (1 W and 10.6 sec pulses) or repaired with interrupted, simple 6-O silk sutures. Group I consisted of seven animals; five with enterotomies repaired by laser welding and two repaired by suturing. Group II consisted of eight animals with each having both laser and suture repairs. Animals were killed and specimens were removed and examined at 1 day, and at 1, 2, and 3 weeks postoperatively to compare the progression of healing. On macroscopic examination the laser welded enterotomies were closed 84% of the time and only 23% had adhesion formation while 90% of sutured repairs were closed and 100% had adhesion formation. Histologic examination of both suture and laser welded enterotomies demonstrated active healing at 1 week with minimal collagen bridging the enterotomies. At 2 and 3 weeks the sutured enterotomies had granulomatous reaction around the sutures while the laser welded enterotomies had minimal inflammatory response and near normal small bowel histology. The tensile strength of the 3-week specimens from both the suture and laser welded enterotomies were 50% of normal bowel. These findings suggest that the laser welding of small bowel enterotomies is comparable in closure and tensile strength to suture repair. The time required to repair the enterotomy is significantly decreased, the procedure is easily performed, and there is a marked decrease in adhesion formation following laser repair.