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1.
Orphanet J Rare Dis ; 14(1): 272, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31779656

RESUMO

BACKGROUND: Congenital Pulmonary Airway Malformation (CPAM) has an estimated prevalence between 0.87 and 1.02/10,000 live births and little is know about their pathogenesis. To improve our knowledge on these rare malformations, we analyzed the cellular origin of the two most frequent CPAM, CPAM types 1 and 2, and compared these malformations with adjacent healthy lung and human fetal lungs. METHODS: We prospectively enrolled 21 infants undergoing surgical resection for CPAM. Human fetal lung samples were collected after termination of pregnancy. Immunohistochemistry and proteomic analysis were performed on laser microdissected samples. RESULTS: CPAM 1 and 2 express mostly bronchial markers, such as cytokeratin 17 (Krt17) or α-smooth muscle actin (ACTA 2). CPAM 1 also expresses alveolar type II epithelial cell markers (SPC). Proteomic analysis on microlaser dissected epithelium confirmed these results and showed distinct protein profiles, CPAM 1 being more heterogeneous and displaying some similarities with fetal bronchi. CONCLUSION: This study provides new insights in CPAM etiology, showing clear distinction between CPAM types 1 and 2, by immunohistochemistry and proteomics. This suggests that CPAM 1 and CPAM 2 might occur at different stages of lung branching. Finally, the comparison between fetal lung structures and CPAMs shows clearly different protein profiles, thereby arguing against a developmental arrest in a localized part of the lung.


Assuntos
Malformação Adenomatoide Cística Congênita do Pulmão/metabolismo , Proteômica/métodos , Actinas/metabolismo , Biomarcadores/metabolismo , Feminino , Feto/metabolismo , Humanos , Imuno-Histoquímica , Queratina-17/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Masculino , Gravidez , Estudos Prospectivos
2.
Histopathology ; 51(1): 87-97, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17593084

RESUMO

AIMS: To investigate the modulation of cellular retinol-binding protein (CRBP)-1 and the desmosomal plaque proteins plakophilin (PKP)-1 and desmoplakin (DP) in correlation with the Ki67+ proliferation index (PI) during the progression of cervical squamous intraepithelial lesions (SIL) to squamous cell carcinoma (SCC). METHODS: Using in situ imaging by brightfield and confocal laser scanning microscopy, the expression of CRBP-1 protein and transcripts, PKP-1, DP and the Ki67 PI were analysed in 38 low-grade (L) SIL, 56 high-grade (H) SIL, 49 SCC, 30 control cervices and 10 human papillomavirus-positive condylomatous lesions. RESULTS: CRBP-1+ cells increased from 11.4% in the normal cervix to 80.3% in LSILs, 92.3% in HSILs and slightly decreased to 78.3% in invasive SCCs (P = 0.0001) in close association with the Ki67 PI (r =0.41; P < 0.0001). PKP-1+ and DP+ cells were correlated (0.32; P < 0.0001) and decreased from normal (81% versus 92.3%) to LSIL (53.1% versus 85.3%), to HSIL (46.4% versus 67.5%) and SCC (35.1% versus 35.9%). The Ki67+ PI was inversely correlated with DP (-0.20, P = 0.0014) and PKP-1 (-0.19, P = 0.015). Condylomata retained low CRBP-1 and high expression of PKP-1 and DP. CONCLUSIONS: The gain of CRBP-1 and the loss of desmosomal proteins occur early in cervical carcinogenesis.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Desmoplaquinas/metabolismo , Antígeno Ki-67/metabolismo , Placofilinas/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Neoplasias do Colo do Útero/metabolismo , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Proliferação de Células , Transformação Celular Neoplásica/genética , Colo do Útero/metabolismo , Colo do Útero/patologia , Condiloma Acuminado/metabolismo , Condiloma Acuminado/patologia , Desmoplaquinas/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Antígeno Ki-67/genética , Placofilinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol , Neoplasias do Colo do Útero/patologia
3.
Handb Exp Pharmacol ; (170): 645-63, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16596818

RESUMO

Proliferation and migration of smooth muscle cells (SMCs) from the media towards the intima are key events in atherosclerosis and restenosis. During these processes, SMC undergo phenotypic modulations leading to SMC dedifferentiation. The identification and characterization of factors controlling these phenotypic changes are crucial in order to prevent the formation of intimal thickening. One of the questions which presently remains open, is to know whether any SMCs of the media are capable of accumulating into the intima or whether only a predisposed medial SMC subpopulation is involved in this process. The latter hypothesis implies that arterial SMCs are phenotypically heterogenous. In this chapter, we will describe the distinct SMC phenotypes identified in arteries of various species, including humans. Their role in the formation of intimal thickening will be discussed.


Assuntos
Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Animais , Apoptose , Diferenciação Celular , Movimento Celular , Proliferação de Células , Citoesqueleto/fisiologia , Humanos
4.
Arterioscler Thromb Vasc Biol ; 21(6): 949-54, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11397702

RESUMO

Cultured arterial smooth muscle cells (SMCs) with distinct phenotypic features have been described by several laboratories; however, it is not presently known whether this phenotypic heterogeneity can be maintained within an in vivo environment. To answer this question, we have seeded into the intima of denuded rat carotid artery 2 SMC populations with well-established distinct biological features, ie, spindle-shaped, not growing in the absence of serum, and well differentiated versus epithelioid, growing in the absence of serum, and relatively undifferentiated, derived from the aortic media of newborn rats (aged 4 days) and old rats (aged >18 months), respectively. We show that these 2 populations maintain their distinct biochemical features (ie, expression of alpha-smooth muscle actin, smooth muscle myosin heavy chains, and cellular retinol binding protein-1) in the in vivo environment. The old rat media-derived SMCs continue to produce cellular retinol binding protein-1 but little alpha-smooth muscle actin and smooth muscle myosin heavy chains, whereas the newborn rat media-derived SMCs continue to express alpha-smooth muscle actin and smooth muscle myosin heavy chains but no cellular retinol binding protein-1. Our results reinforce the notion of arterial SMC phenotypic heterogeneity and suggest that in our model, heterogeneity is controlled genetically and not by the local environment.


Assuntos
Artérias/citologia , Lesões das Artérias Carótidas/cirurgia , Músculo Liso Vascular/transplante , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Arteriosclerose/metabolismo , Arteriosclerose/cirurgia , Lesões das Artérias Carótidas/metabolismo , Células Cultivadas , Masculino , Músculo Liso Vascular/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Ratos , Ratos Endogâmicos F344 , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao Retinol
5.
J Cell Physiol ; 187(3): 315-25, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11319755

RESUMO

We have reported that Cellular Retinol Binding Protein-1 (CRBP-1) is expressed de novo during skin wound healing by a proportion of fibroblastic cells which then differentiate into myofibroblasts and express alpha-smooth muscle actin. In fibroblasts cultured from different tissues we have shown that alpha-smooth muscle actin expression, mainly controlled by Transforming Growth Factor-beta (TGF-beta), is also regulated by retinoic acid and that CRBP-1, known to be a retinoic acid-responsive gene, is modulated by TGF-beta. The aim of the present study has been to investigate the relationships between retinoic acid and TGF-beta in regulating the expression of CRBP-1 and alpha-smooth muscle actin in cultured rat subcutaneous tissue fibroblasts. We have observed that the TGF-beta-induced, but not the retinoic acid-induced, alpha-smooth muscle actin expression is associated with a modulation of endogenous TGF-beta and TGF-beta receptors, suggesting that the action of retinoic acid on alpha-smooth muscle actin expression is not mediated by TGF-beta. The expression of CRBP-1 is regulated at the transcriptional level by TGF-beta and retinoic acid but not synergistically, suggesting a possible common pathway. However, retinoic acid, but not TGF-beta, increases the transcription of a transiently transfected chimeric construct containing the retinoic acid response element of the CRBP-1 promoter, indicating that TGF-beta does not influence CRBP-1 through the retinoic acid pathway. Our results indicate that distinct pathways regulate the genes involved in the appearance and evolution of the myofibroblastic cells. The characterization of these pathways will be helpful for the design of drugs influencing wound healing.


Assuntos
Actinas/biossíntese , Fibroblastos/metabolismo , Proteínas de Ligação ao Retinol/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/metabolismo , Actinas/genética , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia
6.
Circulation ; 103(6): 882-8, 2001 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-11171799

RESUMO

BACKGROUND: To characterize the cells responsible for neointima formation after porcine coronary artery wall injury, we studied the expression of smooth muscle cell (SMC) differentiation markers in 2 models: (1) self-expanding stent implantation resulting in no or little interruption of internal elastic lamina and (2) percutaneous transluminal coronary angioplasty (PTCA) resulting in complete medial rupture and exposure of adventitia to blood components. METHODS AND RESULTS: The expression of alpha-smooth muscle (SM) actin, SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin was investigated by means of immunohistochemistry and Western blots in tissues of the arterial wall collected at different time points and in cell populations cultured from these tissues. The expression of smoothelin, a marker of late SMC differentiation, was used to discriminate between SMCs and myofibroblasts. Both stent- and PTCA-induced neointimal tissues and their cultured cell populations expressed all 4 markers. The adventitial tissue underlying PTCA-induced lesions temporarily expressed alpha-SM actin, desmin, and SM myosin heavy chain isoforms, but not smoothelin. When placed in culture, adventitial cells expressed only alpha-SM actin. CONCLUSIONS: Our results suggest that SMCs are the main components of coronary artery neointima after both self-expanding stent implantation and PTCA. The adventitial reaction observed after PTCA evolves with a chronology independent of that of neointima formation and probably corresponds to a myofibroblastic reaction.


Assuntos
Vasos Coronários/lesões , Músculo Liso Vascular/lesões , Actinas/metabolismo , Angioplastia Coronária com Balão , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Vasos Coronários/fisiologia , Vasos Coronários/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Desmina/metabolismo , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/ultraestrutura , Imuno-Histoquímica , Modelos Animais , Proteínas Musculares/metabolismo , Músculo Liso Vascular/fisiologia , Músculo Liso Vascular/ultraestrutura , Cadeias Pesadas de Miosina/metabolismo , Coloração e Rotulagem , Stents , Suínos , Fatores de Tempo
8.
Invest Ophthalmol Vis Sci ; 41(8): 2336-42, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10892881

RESUMO

PURPOSE: Formation of scarlike epiretinal membranes (ERMs) constitutes potentially the end stage of evolution of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Among various cellular populations, ERMs contain cells with contractile features typical of myofibroblasts. The current study was conducted to investigate the presence of transforming growth factor (TGF)-beta1, TGF-beta receptor II (RII) and ED-A fibronectin (FN), the main inducers of myofibroblastic differentiation in ERMs in PDR and PVR. METHODS: Samples of ERM were obtained from 23 patients during microsurgery for PVR or PDR. Electron microscopy, immunohistochemistry, and confocal microscopy with antibodies recognizing beta-smooth muscle (SM) actin, desmin, TGF-beta1, TGF-beta receptors I and II, and ED-A FN were performed. RESULTS: alpha-SM actin was detected in all ERMs, whereas desmin was present in 50% of the cases. ED-A FN was expressed in all ERMs in close relation with alpha-SM actin-positive myofibroblasts. In addition, TGF-beta1 and TGF-beta R II were always present, TGF-beta RII being expressed in both alpha-SM actin-positive and negative fibroblastic cells. CONCLUSIONS: Myofibroblast accumulation is a key event in ERM-associated traction retinal detachment occurring during PVR and PDR. The current results suggest that the presence of alpha-SM actin-positive myofibroblasts is probably dependent on the concomitant neoexpression of TGF-beta1, TGF-beta RII, and ED-A FN. The results furnish new data on the mechanism of alpha-SM actin stimulation in fibroblasts in a human pathologic setting.


Assuntos
Fibroblastos/metabolismo , Fibronectinas/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Vitreorretinopatia Proliferativa/metabolismo , Actinas/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Membrana Epirretiniana/metabolismo , Membrana Epirretiniana/patologia , Feminino , Fibroblastos/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Vitreorretinopatia Proliferativa/patologia
9.
Int J Radiat Oncol Biol Phys ; 46(3): 661-70, 2000 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-10701746

RESUMO

PURPOSE: Ionizing radiation has been shown to be a powerful inhibitor of neointimal hyperplasia following arterial injury in several animal models of post-percutaneous transluminal coronary angioplasty (post-PTCA) restenosis. This was previously shown to be associated with a reduction in smooth muscle cell (SMC) mitotic activity. This study evaluated the effect of intraarterial beta irradiation on the arterial wall SMC density and apoptosis. METHODS AND MATERIALS: Twenty-five carotid and 7 iliac arteries of hypercholesterolemic New Zealand white rabbits were injured using the Baumgartner technique. The impact of an 18 Gy beta radiation dose administered after balloon injury was studied and compared to a nonirradiated injured control group. The medial SMC density as well as the percentage of apoptotic cells were determined at 8 days, 21 days, and 6 weeks after injury using an automated computer-based software. Apoptotic cells were identified using in situ end-labeling of fragmented DNA. RESULTS: The values for medial apoptosis in control vs. irradiated arteries were: 0.014 +/- 0.023 vs. 0.23 +/- 0.28%, p = NS, at 8 days; 0.012 +/- 0.018 vs. 0.07 +/- 0.07%, p = 0.05, at 21 days; and 0 +/- 0 vs. 0.16 +/- 0.11%, p = 0.03, at 6 weeks. The overall incidence of medial apoptotic cells at all time points was 0.01 +/- 0.017 vs. 0.13 +/- 0.14% in controls and irradiated arteries respectively, p = 0.004. Medial SMC density was significantly decreased in irradiated arteries in comparison with controls (p < 0.01 at all time-points). CONCLUSIONS: Intraarterial beta irradiation stimulates medial SMC apoptosis in balloon-injured arteries. This, together with a decrease in SMC mitotic activity, contributes to a decrease in the arterial wall cellularity.


Assuntos
Apoptose , Músculo Liso Vascular/efeitos da radiação , Animais , Apoptose/genética , Partículas beta , Cateterismo , Constrição Patológica/patologia , Constrição Patológica/fisiopatologia , Constrição Patológica/radioterapia , Fragmentação do DNA , Feminino , Hipercolesterolemia/patologia , Hipercolesterolemia/fisiopatologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Coelhos , Radiobiologia , Fatores de Tempo
10.
Circ Res ; 86(5): 520-5, 2000 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-10720413

RESUMO

We compared the effects of NO donors and cGMP analogues on the growth of aortic smooth muscle cells (SMCs) derived from newborn, adult (aged 3 months), and old (aged 2 years) rats. We found that the NO donor S-nitroso-N-acetylpenicillamine failed to block DNA synthesis in SMCs from old rats but was effective in SMCs from newborn and adult rats. However, cGMP analogues were inhibitory in all 3 SMC types. We demonstrated that in SMCs from old rats, NO was unable to increase the concentration of intracellular cGMP, suggesting that either cGMP synthesis was defective or cGMP degradation was enhanced. Western blot analysis revealed that SMCs from old rats do not express the beta subunit of soluble guanylyl cyclase. To confirm the importance of this observation in vivo, we balloon-injured the carotid arteries of adult and old rats. Whereas soluble guanylyl cyclase was expressed at the same level in the media of injured vessels and uninjured vessels of both groups, its expression in the intimas of old rats was reduced by 70% compared with intimas from adult animals. Furthermore, N(omega)-nitro-L-arginine, an inhibitor of NO synthesis, enhanced the intimal thickening in injured vessels in adult rats but not in old rats. We conclude that the loss of NO responsiveness in aged rats is due to the lack of the beta subunit of soluble guanylyl cyclase, and we speculate that this defect contributes to the enhanced intimal thickening in response to injury in old animals.


Assuntos
Envelhecimento/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Músculo Liso Vascular/enzimologia , Óxido Nítrico/metabolismo , Angioplastia com Balão/efeitos adversos , Animais , Aorta/citologia , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/patologia , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Hiperplasia/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/lesões , Doadores de Óxido Nítrico/farmacologia , Nitroarginina/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Proteínas Quinases/metabolismo , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/fisiologia , Solubilidade , Túnica Íntima/citologia , Túnica Íntima/enzimologia , Túnica Íntima/lesões
11.
Arterioscler Thromb Vasc Biol ; 19(12): 2854-62, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10591661

RESUMO

Vascular smooth muscle cells (SMCs) are important targets for endothelium-derived nitric oxide (NO), but this production is attenuated in injured and diseased arteries and during aging. However, SMCs can produce NO themselves by expressing an inducible form of NO synthase (iNOS) under inflammatory conditions and in the repair process after arterial injury. We examined iNOS expression in SMCs derived from the aortic media of newborn, young adult, and old rats. Our results show that SMCs from newborn rats cannot produce significant amounts of NO on stimulation with interferon-gamma plus lipopolysaccharide or interleukin-1beta. In contrast, SMCs from old rats exhibit markedly enhanced iNOS activity. The difference in iNOS activity between the newborn and the old SMCs was closely correlated with levels of iNOS protein, mRNA, and gene promoter activity. Similarly, intercellular adhesion molecule-1 (ICAM-1) was also expressed more abundantly in the old than in the newborn SMCs in response to cytokines. Both iNOS and ICAM-1 are transcriptionally regulated by nuclear factor kappaB (NF-kappaB). Our data demonstrate an intense transactivation of NF-kappaB in old SMCs on tumor necrosis factor-alpha stimulation but only a weak one in newborn SMCs. The difference in the NF-kappaB activation could be explained by a much faster and more extensive IkappaBalpha degradation in old than in newborn SMCs. These data indicate that the capability to respond to proinflammatory stimuli by activating NF-kappaB differs between SMCs at different stages of development. This results in differential capability to express NF-kappaB-dependent genes such as iNOS and ICAM-1, which could have implications for host defense and the pathogenesis of vascular diseases.


Assuntos
Envelhecimento/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase/genética , Animais , Animais Recém-Nascidos , Aorta Torácica/citologia , Células Cultivadas , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/biossíntese , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia
12.
Circ Res ; 85(1): 99-107, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10400915

RESUMO

Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-beta1 or TGF-beta2, and all-trans-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of alpha-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with alpha-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-beta1 or TGF-beta2 and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis.


Assuntos
Vasos Coronários/citologia , Músculo Liso Vascular/citologia , Animais , Artérias/citologia , Biomarcadores , Diferenciação Celular/fisiologia , Células Cultivadas , Heparina/farmacologia , Suínos , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia
13.
Differentiation ; 65(3): 151-9, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10631812

RESUMO

Vascular smooth muscle cells (SMC) in vivo are highly heterogeneous phenotypically, particularly during development and in the adult during periods of remodeling. Much remains to be learned, however, regarding regulation of the SMC phenotype at the gene level. Here, we studied smooth muscle myosin heavy chain (SMMHC) expression at the transcriptional and mRNA levels in SMC cultured from newborn, adult, and old animals, which express different patterns of differentiation markers. We also examined regulation of SMMHC gene expression by TGF-beta, a cytokine known to be involved in the differentiation process. The activity of SMMHC promoter constructs, the expression of which is smooth-muscle-specific, was greatest in SMC from newborn animals and least in cells from old animals. Thus, differences in the degree of differentiation of SMC from these three sources may at least in part be due to transcriptional events. SMC from the three animal sources each contained mRNAs for the SM-1A and SM-2A tail but not those for the SM-1B and SM-2B head isoforms. Total SMMHC mRNA levels reflected similar differences as found at the transcriptional level. SM-2A mRNA as a proportion of total SMMHC mRNA was greatest in SMC from newborn animals, consistent with their higher degree of differentiation. TGF-beta up-regulated both transcription and mRNA levels but did not change the proportions of SMMHC mRNAs. Though the levels of transcriptional activity and mRNA were widely different in untreated cells, the degree of TGF-beta stimulation was approximately the same in all cases.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/citologia , Cadeias Pesadas de Miosina/biossíntese , Isoformas de Proteínas/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Fatores Etários , Animais , Aorta Torácica , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Músculo Liso Vascular/metabolismo , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica/efeitos dos fármacos
14.
J Cell Biol ; 142(3): 873-81, 1998 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-9700173

RESUMO

Transforming growth factor-beta1 (TGFbeta1), a major promoter of myofibroblast differentiation, induces alpha-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes alpha-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFbeta1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of alpha-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se alpha-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFbeta1-triggered enhancement of alpha-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A-containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFbeta1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.


Assuntos
Diferenciação Celular/fisiologia , Fibroblastos/citologia , Fibronectinas/fisiologia , Músculo Liso/citologia , Fator de Crescimento Transformador beta/fisiologia , Actinas/metabolismo , Animais , Matriz Extracelular/fisiologia , Feminino , Fibronectinas/química , Humanos , Fenótipo , Ratos , Ratos Wistar , Transdução de Sinais
15.
Circ Res ; 82(10): 1086-93, 1998 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9622161

RESUMO

Cultured rat aortic smooth muscle cells (SMCs) exhibit at least 2 phenotypic variants: (1) a spindle-shaped phenotype, obtained from normal adult media, and (2) an epithelioid phenotype, obtained from intimal thickening 15 days after endothelial injury. Both phenotypes can be cloned from each location, with normal media yielding a majority of spindle-shaped clones and intimal thickening yielding a majority of epithelioid clones. These findings suggest that intimal thickening develops essentially from a subpopulation of medial SMCs exhibiting epithelioid features in vitro. Using zymographic and Northern blot analyses, we have studied plasminogen activator (PA) expression by these SMCs. Our results show that epithelioid SMCs, cultured as whole SMC populations or as clones, display higher PA activity than do spindle-shaped SMCs, irrespective of their origin. This is mainly due to differences in the expression of tissue PA and, to a lesser extent, urokinase PA and is accompanied by a decrease in PA inhibitor 1. Tissue PA activity is increased by basic fibroblast growth factor and platelet-derived growth factor-BB, particularly in epithelioid SMCs. Taken together, these results indicate that SMCs are heterogeneous with respect to their proteolytic profile, at least as far as the PA system is concerned. Proteolytic activity of the different SMC populations is modulated by cytokines that play a role in intimal thickening. Our results are in agreement with the suggestion that epithelioid SMCs are mainly responsible for intimal thickening.


Assuntos
Citocinas/farmacologia , Músculo Liso Vascular/metabolismo , Ativadores de Plasminogênio/biossíntese , Animais , Becaplermina , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fenótipo , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Fator de Crescimento Transformador beta/farmacologia , Fatores de Crescimento Transformadores/farmacologia
16.
Int J Biochem Cell Biol ; 29(1): 19-30, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9076938

RESUMO

Following injury, tissue repair involves inflammation, granulation tissue formation and scar constitution. Granulation tissue develops from the connective tissue surrounding the damaged or missing area and contains mainly small vessels, inflammatory cells, fibroblasts and myofibroblasts. As the wound closes and evolves into a scar, there is a striking decrease in cellularity, including disappearance of typical myofibroblasts. The question arises as to what process is responsible for granulation tissue cell disappearance. Our results (in cutaneous wounds) and results of other laboratories (particularly in lungs and kidney) suggest that apoptosis is the mechanism responsible for the evolution of granulation tissue into a scar. During excessive scarring (hypertrophic scar or fibrosis), it is conceivable that the process of apoptosis cannot take place. After experimental endothelial injury in an artery, accumulation of smooth muscle cells participates in the formation of intimal thickening. Apoptotic features have been observed in cells of intimal thickening and also within human atherosclerotic plaques. In the case of atherosclerosis, apoptosis could be detrimental: since smooth muscle cells participate in plaque stability, apoptosis could lead to weakening and rupture of the plaque. These results underline the fact that both increased cell survival or excessive cell death can be associated with pathological disorders. Specific therapies devised to enhance or decrease the susceptibility of individual cell types to apoptosis development could modify the evolution of a variety of human diseases.


Assuntos
Apoptose/fisiologia , Cicatrização/fisiologia , Animais , Vasos Sanguíneos/lesões , Vasos Sanguíneos/patologia , Cicatriz/patologia , Fibrose , Tecido de Granulação/patologia , Humanos , Microscopia Eletrônica
17.
Arterioscler Thromb Vasc Biol ; 16(6): 815-20, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8640410

RESUMO

It is well accepted that smooth muscle cells (SMCs) cultured from normal rat arterial media have different morphological and biological features compared with SMCs cultured from experimental intimal thickening (IT) 15 days after endothelial injury. It is not known, however, whether the phenotypic modulation producing IT cells occurs in any medial SMCs or only in a particular SMC subpopulation. To distinguish among these possibilities, the phenotypic features of SMC clones derived from normal adult media and the IT 15 days after endothelial lesion were analyzed according to morphological appearance, replicative activity in the presence and absence of fetal calf serum, and [3H]thymidine incorporation and motile activity; these features were compared with those of the respective SMC parental populations. Two categories of SMC clones predominated: spindle clones, with morphological features similar to those of the parental population from the normal media, and epithelioid clones, with morphological features similar to those of the IT parental population. Both categories were present among clones produced from normal media and IT; however, spindle was more common among normal media clones, and epithelioid, among IT clones. The behavior in vitro was distinct for each category of clones and did not depend on their origin. Our results are compatible with the possibility that the SMC population of IT in vivo derives mainly from SMCs belonging to the category exhibiting epithelioid features in vitro.


Assuntos
Aorta Torácica/lesões , Endotélio Vascular/lesões , Músculo Liso Vascular/patologia , Animais , Aorta Torácica/patologia , Divisão Celular , Movimento Celular , Células Clonais/ultraestrutura , Citoesqueleto/ultraestrutura , Replicação do DNA , Embolectomia/efeitos adversos , Fenótipo , Ratos , Ratos Wistar
18.
Differentiation ; 60(1): 47-57, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8935928

RESUMO

We demonstrate that self-renewing myoblasts can be identified in the progeny of single human muscle satellite cells (HMSC) in culture. We show, using cytoskeletal proteins and cell size as markers, that self-renewing myoblasts are phenotypically different from other myoblasts, but similar to native HMSC. Native desmin-positive HMSC, cultured as single cells, yielded two major populations of myoblasts, alpha-sarcomeric (alpha-SR)-actin-positive myoblasts and desmin-positive myoblasts. In appropriate culture conditions, alpha-SR-actin-positive myoblasts fused into myotubes, whereas a population of desmin-positive non-fusing myoblasts (NFMB) persisted for weeks among the myotubes. Upon isolation from myotubes, some of the NFMB resumed proliferation and their progeny included fusing and non-fusing myoblasts, with the same cytoskeletal phenotypes as the progeny of native HMSC. This self-renewal cycle could be repeated, yielding four cohorts of myoblasts. The yield of self-renewing cells appeared to decrease with the number of cycles. These results suggest that stem cells are present among NFMB. Moreover, we find that these presumptive stem cells are already segregated during myoblast proliferation. They are small, phenotypically similar to native HMSC, and do not divide unless they are isolated from their sister progeny and cultured alone. Enriched preparations of cells with stem cell-like properties can be obtained from proliferating myoblasts by flow cytometry on the basis of size and nucleocytoplasmic ratio.


Assuntos
Músculo Esquelético/citologia , Células-Tronco/citologia , Adolescente , Adulto , Divisão Celular , Tamanho Celular , Células Cultivadas , Criança , Pré-Escolar , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citometria de Fluxo , Humanos , Lactente , Músculo Esquelético/metabolismo
19.
Lab Invest ; 74(1): 265-78, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8569191

RESUMO

The animal model of hepatic fibrosis induced by bile duct ligation represents an experimental model of human chronic biliary fibrosis. Much attention has been given to the hepatic stellate cell (HSC), or perisinusoidal cell, as the source of the extracellular matrix proteins. However, in the bile duct ligation model, mesenchymal cells other than HSC may be involved in the early stages of fibrosis development. The current study examined, in Sprague-Dawley rats, proliferation in different liver cell subpopulations as well as expression of alpha-smooth muscle (SM) actin and desmin in portal fibroblasts and HSC at 6 hours and 1, 2, 3, and 7 days after bile duct ligation. Kinetics of liver cell proliferation and of phenotypic modulation of portal fibroblasts and HSC (expression of alpha-SM actin and desmin) was evaluated by immunocytochemistry, immunofluorescence, and immunoelectron microscopy using immunogold technique. In sham-operated animals, the evaluation of proliferation in various liver cell subpopulations revealed nonsignificant changes compared with nonoperated rats. alpha-SM actin was detected in vessel walls but was absent in cells of portal tract and parenchyma. Desmin was expressed in vessel walls and in some fibroblastic cells of portal stroma (8.2 cells/unit area) as well as in HSC in acinar Zones 1 and 3 (15.6 cells/unit area and 7.1 cells/unit area, respectively). In bile duct-ligated rats, 24 and 48 hours after ligation, marked proliferations of bile duct epithelial cells (labeling indices 36.8% and 29.5%, respectively) and of periductular fibroblasts (labeling indices 16.7% and 31.0%, respectively) were observed; thereafter, proliferation decreased for both populations (labeling indices at 7 days 12.0% and 11.6%, respectively). HSC proliferation increased gradually until the third day (labeling index 18.6%) and then leveled off. Immunocytochemistry and immunoelectron microscopy revealed a significant number of cells expressing alpha-SM actin 72 hours after bile duct ligation in the stroma adjacent to proliferating ductules. The number of alpha-SM actin-positive cells increased until the seventh day (251.6 cells/unit area). At all times examined, the distribution of alpha-SM actin was restricted to the connective tissue stroma adjacent to proliferating ductules; alpha-SM actin was not expressed in HSC of the lobule. An expansion of desmin expression was noted in fibroblastic cells in stroma surrounding proliferating ductules until 72 hours after bile duct ligation (74.7 cells/unit area) followed by a plateau. At this time, desmin expression increased also in HSC; as in controls, the number of positive cells was greater in Zone 1 (31.8 cells/unit area) than in Zone 3 (18.5 cells/unit area). Double immunofluorescence staining detected by confocal microscopy showed that the majority of portal fibroblastic cells expressing alpha-SM actin was desmin negative 48 hours after bile duct ligation. From 72 hours, portal fibroblastic cells coexpressing alpha-SM actin and desmin appeared, and their proportion increased until 7 days. The present findings indicate that in the early phase of bile duct ligation, there is a marked and transient proliferation of bile duct epithelial cells associated with proliferation of portal periductular fibroblasts, which rapidly express alpha-SM actin. This fibroblastic population may play a dominant role in the early portal fibrosis after bile duct ligation.


Assuntos
Colestase/patologia , Fibroblastos/patologia , Sistema Porta/patologia , Actinas/análise , Animais , Divisão Celular , Colestase/sangue , Colestase/metabolismo , Colágeno/química , Desmina/análise , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Fibrose , Técnica Indireta de Fluorescência para Anticorpo , Técnicas Imunoenzimáticas , Masculino , Microscopia Confocal , Sistema Porta/ultraestrutura , Ratos , Ratos Sprague-Dawley
20.
Differentiation ; 59(4): 259-68, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8575648

RESUMO

We examined whether freshly isolated (native) human muscle satellite cells (HMSC), as well as their proliferating clonal progenies, were heterogeneous. We studied the expression of the cytoskeletal proteins, desmin (DSM), alpha-sarcomeric and alpha-smooth muscle actins (alpha-SR actin, alpha-SM actin), three markers that may be expressed prior to the fusion process. We found that native HMSC constituted a homogeneous population of cells expressing desmin and giving rise to similar clones in vitro. The clonal progeny of HMSC was heterogeneous, including several subpopulations of myoblasts with different cytoskeletal phenotypes, commitment states and fusion abilities. A major subpopulation that expressed both alpha-sarcomeric actin and desmin during the proliferative stage corresponded to a "predifferentiated" population of myoblasts, committed to fusion. Another subpopulation, expressing exclusively desmin, and phenotypically similar to native HMSC, failed to fuse under fusion-promoting conditions and could represent a new generation of HMSC born in culture.


Assuntos
Actinas/análise , Proteínas do Citoesqueleto/análise , Desmina/análise , Heterogeneidade Genética , Proteínas Musculares/análise , Músculo Esquelético/metabolismo , Adolescente , Adulto , Divisão Celular/fisiologia , Criança , Pré-Escolar , Células Clonais , Humanos , Lactente , Músculo Esquelético/citologia , Fenótipo
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