RESUMO
Human bone marrow was harvested by means of iliac crest aspiration and cultured under conditions that promote an osteoblastic phenotype. Human bone marrow aspirates from 30 normal subjects, ages 8-80 years, with no systemic illness, yielded a mean of 92 +/- 65 x 10(6) nucleated cells per 2 ml of aspirate. The prevalence of potential osteoblastic progenitors was estimated by counting the number of alkaline phosphatase-positive colonies. This assay demonstrated a mean of 43 +/- 28 alkaline phosphatase-positive colonies per 10(6) nucleated cells, which was about one per 23,000 nucleated cells. The prevalence of these colonies was positively correlated with the concentration of nucleated cells in the original aspirate (p = 0.014) and was negatively correlated with donor age (p = 0.020). The population of alkaline phosphatase-positive colonies in this model sequentially exhibited markers of the osteoblastic phenotype; essentially all colonies (more than 99%) stained positively for alkaline phosphatase on day 9. Matrix mineralization, which was associated with the synthesis of bone sialoprotein, was demonstrated on day 17 with alizarin red S staining. On day 45, cells that were stimulated with 1,25-dihydroxyvitamin D3 synthesized and secreted osteocalcin at concentrations consistent with known osteoblastic cell lines. This model provides a useful method for the assay of progenitors of connective tissue from human subjects, examination of the effects of aging and selected disease states on this progenitor population, and investigation into the regulation of human osteoblastic differentiation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Osteoblastos/citologia , Adipócitos/citologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/análise , Biópsia por Agulha , Medula Óssea/patologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Núcleo Celular , Células Cultivadas , Criança , Feminino , Células-Tronco Hematopoéticas/enzimologia , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Osteoblastos/enzimologia , Osteocalcina/análise , Osteocalcina/genética , Fenótipo , Fatores Sexuais , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Células Estromais/citologia , Células Estromais/enzimologiaRESUMO
Cystathionine beta-synthase (CBS) deficiency is an inborn error of amino acid metabolism that has pleiotropic manifestations and is commonly called "homocystinuria." The features include skeletal, ocular, and vascular defects, some of which are reminiscent of those found in Marfan syndrome (MFS). Because of the spectrum of clinical effects, the pathogenesis of homocystinuria has long been thought to involve the extracellular matrix (ECM), and the condition has been classified as a heritable disorder of connective tissue. Because of the superficial similarities with MFS, we and others (Pyeritz, in McKusicks Heritable Disorders of Connective Tissue, St. Louis, Mosby-Year Book Inc., 5th ed., pp 137-178, 1993; Pyeritz, in Principles and Practice of Medical Genetics, New York, Churchill Livingstone, 3rd ed., pp 1027-1066, 1997; Mudd, Levy, and Skovby, in The Metabolic and Molecular Bases of Inherited Disease, New York, McGraw-Hill Publishing Co., 7th ed., pp 1279-1327, 1995) have speculated how CBS deficiency might affect fibrillin-1, the protein altered in MFS. For example, the altered plasma concentrations of homocysteine and/or cysteine in patients with CBS deficiency may hinder fibrillin-1 synthesis, deposition, or both. When arterial smooth muscle cells were cultured under conditions of cysteine deficiency, fibrillin-1 deposition into the ECM was greatly diminished as revealed by immunocytochemistry. Excessive homocysteine, in contrast, had little, if any, effect on fibrillin-1 deposition. When cysteine concentrations were returned to normal, the smooth muscle cells began to accumulate a matrix rich in fibrillin-1. Type I collagen, the major matrix component synthesized by these smooth muscle cells, was not reduced by low cysteine concentrations nor high homocysteine concentrations. These results demonstrate that a deficiency of cysteine and subsequent inhibition of fibrillin-1 accumulation in CBS deficient patients may be at least partly responsible for their phenotype, and suggest that maintenance of normal plasma cyst(e)ine levels may be an important therapeutic goal.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Cistationina beta-Sintase/metabolismo , Cisteína/deficiência , Proteínas dos Microfilamentos/metabolismo , Animais , Células Cultivadas , Fibrilina-1 , Fibrilinas , Imuno-Histoquímica , CoelhosRESUMO
Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.
Assuntos
Divisão Celular/fisiologia , Colágeno/biossíntese , Músculo Liso Vascular/metabolismo , Pró-Colágeno/biossíntese , Animais , Células Cultivadas , Hibridização de Ácido Nucleico , Pró-Colágeno/genética , RNA Mensageiro/biossíntese , Coelhos , Tubulina (Proteína)RESUMO
Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Büdy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.
Assuntos
Ceruloplasmina/fisiologia , Cisteína/biossíntese , Cisteína/química , Dissulfetos/química , Homocisteína/análogos & derivados , Homocisteína/química , Homocistina/biossíntese , Albumina Sérica/fisiologia , Ceruloplasmina/química , Ceruloplasmina/farmacologia , Cromatografia Líquida de Alta Pressão , Cobre/química , Cobre/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Biológicos , Oxigênio/metabolismo , Ligação Proteica , Albumina Sérica/química , Albumina Sérica/metabolismo , Compostos de Sulfidrila/química , Fatores de TempoRESUMO
To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.
Assuntos
Colágeno/metabolismo , Elevação dos Membros Posteriores/fisiologia , Músculo Esquelético/metabolismo , Animais , Compostos Azo/metabolismo , Colágeno/genética , Imuno-Histoquímica , Masculino , Músculo Esquelético/citologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Greater than 70% of homocysteine in circulation is protein-bound. An in vitro model system using human plasma has been developed to study mechanisms of protein-bound homocysteine formation and establish the equilibrium binding capacities of plasma for homocysteine. Addition of homocysteine to plasma caused an initial rapid displacement of cysteine and a subsequent increase in protein-bound homocysteine. This rapid reaction was followed by a slower oxygen-dependent reaction forming additional protein-bound homocysteine. To determine the equilibrium binding capacity of plasma proteins for homocysteine, plasma was treated with 0.5-10 mM dl-homocysteine for 4 h at 37 degrees C under aerobic conditions. Under these conditions the equilibrium binding capacity was 4.88 +/- 0.51 and 4.74 +/- 0.68 micromol/g protein for male (n = 10) and female (n = 10) donors, respectively. The mechanism of protein-bound homocysteine formation involves both thiol-disulfide exchange and thiol oxidation reactions. We conclude that plasma proteins have a high capacity for binding homocysteine in vitro.
Assuntos
Proteínas Sanguíneas/metabolismo , Homocisteína/sangue , Feminino , Homocisteína/metabolismo , Humanos , Técnicas In Vitro , Masculino , Consumo de Oxigênio , Ligação Proteica , Fatores de TempoRESUMO
An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds. It is known that albumin combines with cysteine in circulation to form albumin-Cys(34)-S-S-Cys. Studies are now presented to show that the formation of albumin-bound homocysteine proceeds through the generation of an albumin thiolate anion. Incubation of human plasma with l-(35)S-homocysteine results in the association of >90% of the protein-bound (35)S-homocysteine with albumin as shown by nonreduced SDS-polyacrylamide gel electrophoresis. Treatment of the complex with beta-mercaptoethanol results in near quantitative release of the bound l-(35)S-homocysteine, demonstrating that the binding of homocysteine to albumin is through a disulfide bond. Furthermore, using an in vitro model system to study the mechanisms of this disulfide bond formation, we show that homocysteine binds to albumin in two steps. In the first step homocysteine rapidly displaces cysteine from albumin-Cys(34)-S-S-Cys, forming albumin-Cys(34) thiolate anion and homocysteine-cysteine mixed disulfide. In the second step, albumin thiolate anion attacks homocysteine-cysteine mixed disulfide to yield primarily albumin-Cys(34)-S-S-Hcy and to a much lesser extent albumin-Cys(34)-S-S-Cys. The results clearly suggest that when reduced homocysteine enters circulation, it attacks albumin-Cys(34)-S-S-Cys to form albumin-Cys(34) thiolate anion, which in turn, reacts with homocysteine-cysteine mixed disulfide or homocystine to form albumin-bound homocysteine.
Assuntos
Albuminas/química , Ânions , Homocisteína/química , Albumina Sérica/química , Albuminas/metabolismo , Cromatografia Líquida de Alta Pressão , Cisteína/química , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mercaptoetanol/farmacologia , Modelos Químicos , Ácido Pentético/farmacologia , Ligação Proteica , Conformação Proteica , Albumina Sérica/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: We tested the hypothesis that the independent risk factor for atherosclerosis of increased plasma homocysteine concentration is associated with the pregnancy syndrome of preeclampsia. We further hypothesized that increased plasma homocysteine concentration during pregnancy may advance endothelial dysfunction in preeclampsia by promoting oxidative stress. STUDY DESIGN: Antepartum blood samples were collected >/=6 hours after the last meal from 33 women with normal, uncomplicated pregnancies and from 21 women with preeclampsia. These plasma samples were analyzed for concentrations of total homocysteine; folate; triglycerides; creatinine; a marker of endothelial activation, cellular fibronectin; and a marker of oxidative stress, malondialdehyde. RESULTS: The mean value of total plasma homocysteine in preeclampsia was significantly higher than that observed in normal pregnancy (P <. 04). Similarly, plasma malondialdehyde (P <.001), triglyceride (P <. 001), and cellular fibronectin (P <.006) concentrations were also greater in women with preeclampsia than in control subjects. However, no differences were observed between women with preeclampsia and control subjects in folate (P =.97) or creatinine (P =.28) concentrations. Homocysteine concentration did not correlate with plasma creatinine (P =.61), malondialdehyde (P =.32), or triglyceride (P =.89) concentrations. However, cellular fibronectin concentration correlated positively with homocysteine concentration in both women with preeclampsia and control subjects (r = 0.87, P <. 0001, and r = 0.50, P <.004, respectively), and folate concentrations were weakly but negatively correlated with homocysteine values (P =.03, r = 0.32). CONCLUSIONS: Total plasma homocysteine concentration is increased in preeclampsia and is significantly correlated with cellular fibronectin concentration, suggesting that homocysteine plays a role in promoting endothelial dysfunction in preeclampsia. Furthermore, despite the use of pregnancy multivitamins and no indications of overt folate deficiency in this subject population, homocysteine concentration weakly and negatively correlates with plasma folate concentration.