RESUMO
Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.
Assuntos
Condrócitos/efeitos dos fármacos , Fator 9 de Crescimento de Fibroblastos/farmacologia , Lâmina de Crescimento/citologia , Lâmina de Crescimento/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Sialoglicoproteínas/metabolismo , Regulação para Cima/efeitos dos fármacos , Acondroplasia/genética , Acondroplasia/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galinhas , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicina/genética , Glicina/metabolismo , Lâmina de Crescimento/metabolismo , Camundongos , Mutação/genética , Osteopontina , Ratos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.
Assuntos
Desenvolvimento Ósseo/fisiologia , Remodelação Óssea/fisiologia , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Calcificação Fisiológica/fisiologia , Mecanotransdução Celular/fisiologia , Neovascularização Fisiológica/fisiologia , Suporte de Carga/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Animais Recém-Nascidos , Osso e Ossos/irrigação sanguínea , Diferenciação Celular/fisiologia , Galinhas , Fêmur/irrigação sanguínea , Fêmur/citologia , Fêmur/crescimento & desenvolvimento , Lâmina de Crescimento/citologia , Lâmina de Crescimento/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Tíbia/irrigação sanguínea , Tíbia/citologia , Tíbia/crescimento & desenvolvimentoRESUMO
The effect of halofuginone (a plant alkaloid) on collagen alpha 1(I) gene expression and collagen synthesis was evaluated in human skin fibroblasts from patients with chronic graft-versus-host disease (cGvHD) or scleroderma and from a normal individual. Halofuginone caused a dose-dependent inhibition in collagen alpha 1(I) gene expression and collagen synthesis in all cultures tested, the cGvHD fibroblasts being the least sensitive. In normal and scleroderma fibroblasts, concentrations of halofuginone as low as 10(-10) M and 10(-9) M were sufficient to cause a significant reduction in collagen alpha 1(I) gene expression and collagen synthesis, respectively. In addition, halofuginone also inhibited the transforming growth factor beta-induced collagen synthesis. Three days after halofuginone removal, collagen gene expression returned to control levels. The reduction of collagen mRNA transcript levels by halofuginone appeared to be dependent on new protein synthesis because simultaneous treatment of fibroblasts with protein synthesis inhibitors prevents the suppressive effect of halofuginone on collagen alpha 1(I) mRNA gene expression. The ability of extremely low concentrations of halofuginone to inhibit collagen alpha 1(I) synthesis specifically and transiently at the transcriptional level suggests that this material may be an important tool for studying collagen alpha 1(I) gene regulation and may be used as a novel and promising antifibrotic therapy.
Assuntos
Coccidiostáticos/farmacologia , Colágeno/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Quinazolinas/farmacologia , Adulto , Colágeno/biossíntese , Relação Dose-Resposta a Droga , Feminino , Humanos , Pessoa de Meia-Idade , Piperidinas , Quinazolinonas , Fatores de TempoRESUMO
The effect of dermal application of halofuginone-an inhibitor of collagen type I synthesis-on skin collagen and collagen alpha1(I) gene expression in an animal model of scleroderma and chronic graft versus host disease (cGvHD) was evaluated. Halofuginone-containing cream was applied on the tight-skin mouse (Tsk) and skin biopsies were taken for collagen staining by sirius red and for collagen alpha1(I) gene expression by in situ hybridization. In addition, cell proliferation was evaluated by immunostaining for proliferation cell nuclear antigen (PCNA) alone or in combination with collagen alpha1(I) probe. The number of mast cells was assessed by toluidine blue. Dermal application of halofuginone (0.01%) for 60 days was as good as systemic administration (1 microg/mouse/day) in reducing collagen alpha1(I) gene expression in skin biopsy and almost as good in reducing skin width. Halofuginone was stable and effective only at acidic pH. The effect of halofuginone (0.03%) was time-dependent. After 40 days of daily treatment, a significant reduction in the collagen alpha1(I) gene expression was observed and further decrease was observed after 60 days. The reduction in collagen alpha1(I) gene expression and the reduction in the proliferation of dermal fibroblasts probably occur in the same subset of cells. No effect of halofuginone on the proliferation of keratinocytes or on mast cell number was observed. These results suggest that target-oriented application of halofuginone may become a novel therapy for fibrotic disorders in general and for scleroderma in particular.
Assuntos
Fibrose/tratamento farmacológico , Inibidores da Síntese de Proteínas/uso terapêutico , Quinazolinas/uso terapêutico , Administração Tópica , Animais , Divisão Celular/efeitos dos fármacos , Colágeno/biossíntese , Colágeno/efeitos dos fármacos , Modelos Animais de Doenças , Fibrose/patologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Camundongos , Piperidinas , Quinazolinonas , Pele/efeitos dos fármacos , Pele/metabolismoRESUMO
PTH/PTHrP receptor gene expression was evaluated in situ in avian epiphyseal growth plates taken from normal, rachitic and tibial dyschondroplasia (TD) afflicted chicks induced by thiram or by genetic selection. In the normal growth plates, PTH/PTHrP receptor gene expression was localized to the maturation zone as demonstrated by the expression of collagen type II (col II), osteopontin (OPN) genes and alkaline phosphatase activity (AP). In TD, either induced by thiram or by genetic selection, normal levels of PTH/PTHrP receptor gene expression were observed up to 21 days post-hatch. In rickets, on the other hand, no PTH/PTHrP receptor gene expression was observed in the growth plate from day 8 of a vitamin D-deficient diet. In cultured chondrocytes, PTH caused time-dependent down-regulation of its own receptor. These results suggest that alterations in the PTH/PTHrP receptor gene expression are associated with rickets but not with TD. The reduction in the PTH/PTHrP receptor gene expression in rickets may be due to the high plasma levels of PTH.
Assuntos
Galinhas , Lâmina de Crescimento/metabolismo , Osteocondrodisplasias/veterinária , Doenças das Aves Domésticas/genética , Receptores de Hormônios Paratireóideos/genética , Raquitismo/veterinária , Animais , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Lâmina de Crescimento/patologia , Hibridização In Situ , Masculino , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo , Raquitismo/genética , Raquitismo/metabolismo , Tíbia/metabolismo , Tíbia/patologiaRESUMO
The role of heat shock proteins (HSP) in the protection of cells from heat stress is well established. However, very little is known about their contribution to thermotolerance in the complexity of a whole homeotherm animal. Here we report on the analysis of protein synthesis in lung and heart muscle tissues of broiler chickens following exposure to high ambient temperature. Half of the flock was treated by an early age exposure to heat (conditioning), to improve thermotolerance. In contrast to what has been expected, lower levels of HSP induction was observed in the treated chickens. We suggest that 1) the induction of HSP in the heart and lung tissues of the whole animal correlates with the body temperature and 2) HSP response does not represent a part of the long-term mechanism that is evoked by the early age conditioning.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Temperatura Corporal/fisiologia , Galinhas/fisiologia , Proteínas de Choque Térmico/biossíntese , Envelhecimento/metabolismo , Animais , Autorradiografia/veterinária , Galinhas/genética , Galinhas/metabolismo , Eletroforese em Gel de Poliacrilamida/veterinária , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/genética , Temperatura Alta/efeitos adversos , Pulmão/metabolismo , Masculino , Miocárdio/metabolismo , Doenças das Aves Domésticas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/metabolismo , Estresse Fisiológico/veterinária , Fatores de TempoRESUMO
Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.
Assuntos
Condrócitos/fisiologia , Lâmina de Crescimento/enzimologia , Metaloproteinases da Matriz/metabolismo , Osteocondrodisplasias/veterinária , Doenças das Aves Domésticas/enzimologia , Raquitismo/veterinária , Animais , Galinhas , Condrócitos/metabolismo , Condrócitos/patologia , Regulação Enzimológica da Expressão Gênica , Lâmina de Crescimento/patologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/patologia , Osteogênese , Doenças das Aves Domésticas/patologia , Distribuição Aleatória , Raquitismo/enzimologia , Raquitismo/patologiaRESUMO
Vertebrate long bones are formed by endochondral ossification, a process accompanied by changes in extracellular matrix synthesis and remodeling, performed mainly by the matrix metalloproteinases (MMP). The temporal/spatial expression patterns of 5 members of the MMP family known to be important for endochondral ossification were studied, for the first time, in the turkey growth plate during embryonic and juvenile stages. The expression of MMP-2 was detected in the proliferative zone, MMP-3, MMP-9, and MMP-13 in cells lining the blood vessels; MMP-13 was also detected in hypertrophic chondrocytes. The MMP-16 expression was detected in the reserve zone of the growth plate. These results present a detailed survey of turkey MMP, serving as a data source (atlas) for further studies in this subject.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Metaloproteinases da Matriz/metabolismo , Osteogênese/fisiologia , Perus/crescimento & desenvolvimento , Perus/metabolismo , Animais , Lâmina de Crescimento/enzimologia , Membro Posterior , Transporte Proteico , Perus/embriologiaRESUMO
Hepatic fibrosis is associated with the activation of stellate cells (HSCs), the major source of extracellular matrix (ECM) proteins. Transforming growth factor-beta (TGF-beta), signaling via Smad3, is the most profibrogenic cytokine and the major promoter of ECM synthesis. Halofuginone, an inhibitor of liver fibrosis, inhibits TGF-beta-dependent Smad3 phosphorylation in human HSCs in culture. We have used transcriptional profiling to evaluate the effect of halofuginone on gene expression during the progression of thioacetamide (TAA)-induced liver fibrosis in the rat and have focused on genes that are associated with TGF-beta. TAA treatment causes alterations in the expression of 7% of liver genes. Halofuginone treatment prevents the changes in the expression of 41% of these genes and results in the inhibition of HSC activation and collagen synthesis. During the early stages of the disease, halofuginone affects genes involved in alcohol, lipid, protein, and phosphate metabolism and cell adhesion and, at later stages, in the cell cycle (cell development, differentiation, cell proliferation, and apoptosis). The activation of TGF-beta-dependent genes, such as tartrate-resistant acid phosphatase, its putative substrate osteopontin, stellate cell activation-association protein, and fibrillin-1, during chemically induced fibrosis is prevented by halofuginone. This study thus highlights the role of TGF-beta signaling in liver fibrosis and especially its potential for pharmacological intervention. Halofuginone, which has demonstrated efficacy and tolerance in animals and humans, could become an effective and novel therapy for liver fibrosis.
Assuntos
Antineoplásicos/farmacologia , Perfilação da Expressão Gênica , Cirrose Hepática/genética , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Animais , Análise por Conglomerados , Citoglobina , Progressão da Doença , Fibrilina-1 , Fibrilinas , Regulação da Expressão Gênica/efeitos dos fármacos , Globinas/genética , Globinas/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato , TioacetamidaRESUMO
Hepatic fibrosis is associated with activation of hepatic stellate cells (HSC), the major source of the extracellular matrix (ECM) proteins. The predominant ECM protein synthesized by the HSC is collagen type I. We evaluated the effect of halofuginone-an inhibitor of collagen synthesis-on thioacetamide (TAA)-induced liver fibrosis in rats. In the control rats the HSC did not express smooth muscle actin, collagen type I gene, or tissue inhibitor of metalloproteinases-2 (TIMP-2), suggesting that they were in their quiescent state. When treated with TAA, the livers displayed large fibrous septa, which were populated by smooth muscle actin-positive cells expressing high levels of the collagen alpha1(I) gene and containing high levels of TIMP-2, all of which are characteristic of advanced fibrosis. Halofuginone given orally before fibrosis induction prevented the activation of most of the stellate cells and the remaining cells expressed low levels of collagen alpha1(I) gene, resulting in low levels of collagen. The level of TIMP-2 was almost the same as in the control livers. When given to rats with established fibrosis, halofuginone caused almost complete resolution of the fibrotic condition. The levels of collagen, collagen alpha1(I) gene expression, TIMP-2 content, and smooth muscle actin-positive cells were as in the control rats. Halofuginone inhibited the proliferation of other cell types of the fibrotic liver in vivo and inhibited collagen production and collagen alpha1(I) gene expression in the SV40-immortalized rat HSC-T6 cells in vitro. These results suggest that halofuginone may become an effective and novel mode of therapy in the treatment of liver fibrosis.
Assuntos
Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/prevenção & controle , Inibidores da Síntese de Proteínas/uso terapêutico , Quinazolinas/uso terapêutico , Animais , Divisão Celular/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Piperidinas , Quinazolinonas , Ratos , Ratos Wistar , TioacetamidaRESUMO
The avian eggshell gland (ESG) is a tissue specialized in transporting the Ca(2+) required for eggshell formation and represents a unique biological system in which the calcification process takes place in a circadian fashion. With the use of RNA fingerprinting, a set of genes differentially induced at the time of calcification was detected, one of which was identified as the alpha(1)-subunit of Na(+)-K(+)-ATPase. The gene was expressed in a circadian manner in both cell types populating the ESG, but in different temporal patterns, suggesting distinct mechanisms of regulation. Ca(2+) flux and mechanical strain were found to regulate gene expression in the inner glandular epithelium and the pseudostratified epithelium facing the lumen, respectively. Mechanical strain also affected gene expression in cell layers facing the lumen in other parts of the oviduct. Only the alpha(1)-isoform, not the alpha(2)- or alpha(3)-isoform, of Na(+)-K(+)-ATPase was expressed in the ESG. In summary, we demonstrate that the alpha(1)-subunit Na(+)-K(+)-ATPase gene is expressed in different epithelial cell types in the ESG and is regulated by various mechanisms, which may reflect the disparity in the physiological roles of the cells in the process of eggshell formation.
Assuntos
Casca de Ovo/metabolismo , Regulação da Expressão Gênica , Oviductos/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Calcificação Fisiológica/genética , Cálcio/metabolismo , Galinhas , Ritmo Circadiano/genética , Casca de Ovo/crescimento & desenvolvimento , Epitélio/metabolismo , Feminino , Perfilação da Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Especificidade de Órgãos/genética , Subunidades Proteicas , RNA/metabolismo , Estresse MecânicoRESUMO
PURPOSE: Urethral strictures are narrowing of the urethra caused by fibrosis due to excessive collagen production in response to an insult. We evaluated the effects of halofuginone, a potent inhibitor of collagen alpha1(I) gene expression, on experimentally induced urethral strictures in vivo and on rat urethral fibroblasts in vitro. MATERIALS AND METHODS: Applying coagulation current to the male rat urethra produced urethral strictures. Halofuginone was given to the animals for 7 days, starting on the day of stricture formation, either orally at 1 and 5 ppm in the diet or by injection of 0.03% halofuginone solution into the urethra. All rats were sacrificed on day 21. Collagen alpha1(I) gene expression was evaluated by in situ hybridization, collagen content by sirius red staining and urethral morphology by urethrogram. RESULTS: Coagulation current produced reproducible strictures with a typical urethrogram appearance, which were associated with increases in collagen alpha1(I) gene expression and collagen content at the stricture site. Halofuginone injected into the urethra or orally at 5 ppm normalized the urethrogram and prevented increases in collagen alpha1(I) gene expression and collagen content. Halofuginone at a concentration of 10-8 M. inhibited the collagen secreted by fibroblasts derived from the rat male urethra, which was due to inhibition of the collagen alpha1(I) gene expression. CONCLUSIONS: Halofuginone prevented stricture formation and may become an important mode of therapy in the prevention of restenosis during urethral stricture formation.
Assuntos
Colágeno/biossíntese , Inibidores da Síntese de Proteínas/uso terapêutico , Quinazolinas/uso terapêutico , Estreitamento Uretral/prevenção & controle , Animais , Modelos Animais de Doenças , Imuno-Histoquímica , Hibridização In Situ , Masculino , Piperidinas , Quinazolinonas , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: The objective of this study was to evaluate the effects of halofuginone-a specific inhibitor of collagen type I synthesis-in preventing uterine horn adhesion formation in rats. STUDY DESIGN: Adhesions were induced by scraping the rat uterine horns until capillary bleeding occurred. Halofuginone was either injected intraperitoneally or administered orally. The number and severity of the adhesions were scored. Collagen alpha1(I) gene expression was evaluated by in situ hybridization; total collagen was estimated by sirius red staining. Collagen synthesis in response to halofuginone was evaluated in cells cultured from the adhesions. RESULTS: Regardless of the administration procedure, halofuginone reduced significantly the number and severity of the adhesions in a dose-dependent manner. Halofuginone prevented the increase in collagen alpha1(I) gene expression observed in the rats that underwent this procedure, thus affecting only the newly synthesized collagen but not the resident collagen. In cells derived from rat uterine horn adhesions, halofuginone induced dose-dependent inhibition of collagen synthesis. CONCLUSIONS: Upregulation of collagen synthesis appears to play a critical role in the pathophysiologic mechanism of adhesion formation. Halofuginone could be used as an important means of understanding the role of collagen in adhesion formation and might become a novel and promising antifibrotic agent for preventing adhesion formation after pelvic surgery.
Assuntos
Colágeno/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Quinazolinas/farmacologia , Doenças Uterinas/prevenção & controle , Administração Oral , Animais , Colágeno/biossíntese , Colágeno/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Injeções Intraperitoneais , Piperidinas , Inibidores da Síntese de Proteínas/uso terapêutico , Quinazolinas/uso terapêutico , Quinazolinonas , Ratos , Ratos Sprague-Dawley , Aderências Teciduais/prevenção & controle , Doenças Uterinas/patologiaRESUMO
Proliferation of vascular smooth muscle cells (SMCs) and accumulation of extracellular matrix (ECM) components within the arterial wall in response to local injury are important etiologic factors in vascular proliferative disorders such as arteriosclerosis and restenosis after angioplasty. Fibrillar and nonfibrillar collagens are major constituents of the ECM that modulate cell shape and proliferative responses and thereby contribute to the pathogenesis of intimal hyperplasia. Halofuginone, an anticoccidial quinoazolinone derivative, inhibits collagen type I gene expression. We investigated the effect of halofuginone on (1) proliferation of bovine aortic endothelial cells and SMCs derived from the same specimen and maintained in vitro, (2) ECM deposition and collagen type I synthesis and gene expression, and (3) injury-induced intimal hyperplasia in vivo. DNA synthesis and proliferation of vascular SMCs in response to serum or basic fibroblast growth factor were abrogated in the presence of as little as 0.1 microgram/mL halofuginone; this inhibition was reversible upon removal of the compound. Under the same conditions, halofuginone exerted a relatively small antiproliferative effect on the respective vascular endothelial cells. Halofuginone also inhibited the synthesis and deposition of ECM components by vascular SMCs as indicated both by a substantial reduction in the amount of sulfated proteoglycans and collagen type I synthesis and gene expression. Local administration of halofuginone in the rabbit ear model of crush injury-induced arterial intimal hyperplasia resulted in a 50% reduction in intimal thickening as measured by a morphometric analysis of the neointima/media ratio. The differential inhibitory effect of halofuginone on vascular SMCs versus endothelial cells, its inhibition of ECM deposition and collagen type I synthesis, and its ability to attenuate injury-induced intimal hyperplasia may place halofuginone alone or in combination with other antiproliferative compounds as a potential candidate for prevention of arterial stenosis and accelerated atherosclerosis.
Assuntos
Artérias/patologia , Colágeno/biossíntese , Músculo Liso Vascular/efeitos dos fármacos , Quinazolinas/farmacologia , Túnica Íntima/patologia , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/efeitos dos fármacos , Humanos , Hiperplasia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Piperidinas , Quinazolinonas , Coelhos , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/metabolismoRESUMO
BACKGROUND/AIMS: Hepatic cirrhosis is characterized by excessive deposition of collagen, resulting from an increase in type I collagen gene transcription. We evaluated the effect of halofuginone-a specific inhibitor of collagen type alpha 1(I) gene expression-on dimethylnitrosamine (DMN)-induced liver fibrosis/cirrhosis in rats. METHODS: Fibrosis was induced by intraperitoneal injection of DMN. Halofuginone (5 mg/kg) was added to the diet. Collagen was stained with Sirius red and collagen alpha 1(I) gene expression was evaluated by in situ hybridization. RESULTS: In control rats, a low level of collagen alpha 1(I) gene expression was observed. A high dose of DMN (1%) caused severe fibrosis, as indicated by induction of collagen alpha 1(I) gene expression and increased liver collagen content. Addition of halofuginone before the onset of fibrosis, almost completely prevented the increase in collagen type I gene expression and resulted in lower liver collagen content. Moreover, halofuginone partially prevented the marked decrease in liver weight and reduced the mortality rate. At a lower dose of DMN (0.25%), which causes mild fibrosis, halofuginone prevented the increase in collagen alpha 1(I) gene expression, prevented the increase in liver collagen deposition and reduced plasma alkaline phosphatase activity, all of which are characteristic of liver fibrosis/ cirrhosis. CONCLUSIONS: These results suggest that halofuginone can be used as an important tool to understand the regulation of the collagen alpha 1(I) gene and may become a novel and promising antifibrotic agent for liver fibrosis/ cirrhosis.
Assuntos
Colágeno/antagonistas & inibidores , Dimetilnitrosamina , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/prevenção & controle , Inibidores da Síntese de Proteínas/farmacologia , Quinazolinas/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Colágeno/genética , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Injeções Intraperitoneais , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática Experimental/patologia , Masculino , Piperidinas , Quinazolinonas , Ratos , Ratos Sprague-DawleyRESUMO
Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion-which was significantly enlarged-was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy.
Assuntos
Colágeno/biossíntese , Regulação da Expressão Gênica , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Sialoglicoproteínas/biossíntese , Tíbia/patologia , Animais , Diferenciação Celular , Galinhas , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno/genética , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocondrodisplasias/patologia , Osteocondrodisplasias/fisiopatologia , Osteopontina , Sialoglicoproteínas/genética , Tíbia/fisiopatologiaRESUMO
OBJECTIVE: To evaluate the effects of halofuginone, a specific inhibitor of collagen type I synthesis, on the postoperative formation of abdominal adhesions in rats. SUMMARY BACKGROUND DATA: Postoperative adhesions remain the leading cause of small bowel obstruction in the Western world. Surgical trauma causes the release of a serosanguineous exudate that forms a fibrinous bridge between two organs. This becomes ingrown with fibroblasts, and subsequent collagen deposition leads to the formation of a permanent adhesion. Most of the drugs used have been clinically ineffective, and none has been specific to a particular extracellular matrix molecule. Therefore, there are serious concerns about the toxic consequences of interfering with the biosynthesis of other collagens, other matrix proteins, or vital collagen-like molecules. METHODS: Adhesions were induced by scraping the cecum until capillary bleeding occurred. The adhesions were scored 21 days later. Halofuginone was either injected intraperitoneally (1 microg/25 g body weight) every day, starting on the day of operation, or added orally at concentrations of 5 or 10 mg/kg, starting 4 days before the operation. Collagen alpha1(I) gene expression was evaluated by in situ hybridization, total collagen was estimated by Sirius red staining, and collagen type III was detected by immunohistochemistry. RESULTS: The adhesions formed between the intestinal walls were composed of collagen and were populated with cells expressing the collagen alpha1(I) gene. Regardless of the administration procedure, halofuginone significantly reduced the number and severity of the adhesions. Halofuginone prevented the increase in collagen alpha1(I) gene expression observed in the operated rats, thus reducing collagen content to the control level. In fibroblasts derived from abdominal adhesions, halofuginone induced dose-dependent inhibition of collagen alpha1(I) gene expression and collagen synthesis. Collagen type III levels were not altered by adhesion induction or by halofuginone treatment. CONCLUSIONS: Upregulation of collagen synthesis appears to have a critical role in the pathophysiology of postoperative adhesions. Halofuginone, an inhibitor of collagen type I synthesis, could be used as an important tool in understanding the role of collagen in adhesion formation, and it may become a novel and promising antifibrotic agent for preventing postoperative adhesion formation.
Assuntos
Colágeno/antagonistas & inibidores , Complicações Pós-Operatórias/prevenção & controle , Inibidores da Síntese de Proteínas/uso terapêutico , Quinazolinas/uso terapêutico , Aderências Teciduais/prevenção & controle , Abdome , Animais , Colágeno/biossíntese , Histocitoquímica , Hibridização In Situ , Piperidinas , Quinazolinonas , Ratos , Ratos Sprague-Dawley , Aderências Teciduais/patologiaRESUMO
Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.
Assuntos
Matriz Extracelular/metabolismo , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Quinazolinas/farmacologia , Animais , Radioisótopos de Carbono , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Colágeno/genética , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Piperidinas , Prolina/metabolismo , Prolina/farmacologia , Quinazolinonas , Ratos , Ratos Wistar , Sulfatos/metabolismo , Sulfatos/farmacologia , TrítioRESUMO
Calcium-sensing receptor (CaR) gene expression and parathyroid hormone (PTH) content were evaluated in situ in chicken parathyroid glands (PG) in relation to changes in plasma calcium. The CaR gene is expressed by the parathyroid chief cells, the same cells that store and secrete PTH. An increase in plasma calcium, achieved by repletion of vitamin D-deficient chicks with a normal diet, by PTH injection, or during eggshell formation, increased the expression of the CaR gene. Low plasma calcium concentration in vitamin D-deficient chicks or in layers, before or after eggshell formation, was associated with decrease in CaR gene expression in the PG. The level of CaR gene expression was inversely correlated with the PTH content of the PG. The results of this study demonstrate for the first time that, in contrast to mammals, the CaR gene expression in the PG of the chicken is inversely associated with changes in plasma calcium.