Refining retinoic acid stimulation for osteogenic differentiation of murine adipose-derived adult stromal cells

Murine adipose-derived adult stromal cells (ADAS) seeded onto appropriate scaffolds and pre-incubated with retinoic acid have been shown to generate in vivo bone rapidly. Prompt resorption ensues, however, as a result of osteoclastogenesis, likely secondary to retinoic acid carryover. In this study, we determined the effects of abbreviated retinoic acid exposure on ADAS osteogenic differentiation. Histological staining and gene expression analysis revealed that longer retinoic acid exposure resulted in better in vitro bone differentiation. However, significant osteogenesis was observed in ADAS after just 15 days of retinoic acid supplementation, suggesting that continual culture with retinoic acid is unnecessary for initiation of the osteogenic program. This was confirmed using ADAS pre-incubated in monolayer with an abbreviated 15 days of retinoic acid exposure before implantation into critical-sized calvarial defects. Similar rates of regeneration were observed between ADAS exposed to for 15 days or for a full 25-day course of retinoic acid before defect repair. Furthermore, by limiting retinoic acid exposure to ADAS in monolayer without scaffold, accelerated bone formation was observed without concomitant osteoclastic resorption. These data suggest that skeletal regeneration may be improved by modulating retinoic acid exposure before implantation, markedly accelerating the repair of bone defects using ADAS.     

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod

Retinoic acid, when administered to pregnant mice on d 11·0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4 , a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8 , which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh , nor of its receptor and downstream target gene Ptc , suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4-induced skeletogenic mesenchymal cell proliferation.     

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod

Retinoic acid, when administered to pregnant mice on d 11·0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4, a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8, which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh, nor of its receptor and downstream target gene Ptc, suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4‐induced skeletogenic mesenchymal cell proliferation.     

维甲酸对树突状细胞增殖及细胞骨架微丝的调节作用

目的观察药理剂量的全反式维甲酸对人单核细胞来源的树突状细胞增殖及细胞骨架微丝的调节作用。方法 rhGM-CSF1000U/mL、IL-4500U/mL诱导人外周血单核细胞向树突状细胞分化,在细胞分化过程中加入0.1μmol/L全反式维甲酸,相差显微镜下观察细胞的形态变化及集落形成,活细胞计数和MTT检测细胞的增殖。成熟树突状细胞经全反式维甲酸处理24h后,用FITC标记的鬼笔环肽检测细胞骨架微丝的形态和分布。结果细胞因子诱导的外周血单核细胞来源的树突状细胞具有典型的树枝状结构。在树突状细胞的分化过程中,全反式维甲酸作用组的树突状细胞的产量明显低于对照组(P0.05)。已经分化成熟的树突状细胞经维甲酸处理后细胞骨架微丝出现重排和分布不均。结论药理剂量的全反式维甲酸抑制树突状细胞的增殖和分化,并可能通过细胞骨架调节树突状细胞的功能。     

Adjuvant activity of all-trans-retinoic acid in C57Bl/6 mice.

The effects of all-trans-retinoic acid were investigated on the immune responses in C57Bl/6 mice after daily oral administration for one week. In selected experiments the immunosuppressive chemicals, cyclophosphamide and cyclosporin A were used in conjunction with retinoic acid. Retinoic acid stimulated the production of antibodies against sheep red blood cells and DNP-Ficoll; however, retinoic acid did not reverse the depression caused by immunosuppressive chemicals. In non-immunized animals retinoic acid stimulated the production of IL-1 but not of IL-2. The mitogenic responses of splenocytes against concanavalin A, phytohemagglutinin and pokeweed mitogen were depressed after the retinoic acid treatment; those against lipopolysaccharide were not influenced. Treatment with retinoic acid did not alter the mixed leukocyte responses but increased the activity of NK cells. Results indicate that retinoic acid may act as an adjuvant via activating macrophages, however, retinoic acid cannot reverse the immunosuppression induced by potent chemicals.     

全反式维甲酸及胆酸钠对卵巢上皮性癌荷瘤裸鼠干预的实验研究

目的 探讨全反式维甲酸及胆酸钠对裸鼠卵巢上皮性癌动物模型干预后的正、副作用。方法 以CAOV3细胞株皮下接种BALB／C裸鼠，分组后分别给予不同的全反式维甲酸及胆酸钠方案用灌胃针给药进行干预，统一处死后留取裸鼠血、肿瘤及各重要脏器组织进行光镜、电镜及免疫组织化学检查，并于给药期间观察裸鼠体重及瘤体变化。结果 接种肿瘤细胞后早期行全反式维甲酸及胆酸钠干预，裸鼠最终肿瘤成瘤率低，即使成瘤需时也延长，肿瘤生长速度明显减慢；接种肿瘤细胞待成瘤后给予裸鼠行全反式维甲酸及胆酸钠干预，两组药物也有抑制肿瘤生长、诱导肿瘤细胞分化作用，而对裸鼠血相无明显抑制作用，对重要脏器未见明显形态学改变。结论 全反式维甲酸及胆酸钠经灌胃针用药后对荷瘤裸鼠的肿瘤确有抑制作用，并以早期用药为佳，对荷瘤裸鼠重要脏器无明显影响。     

Inhibition of antibody-dependent cellular cytotoxicity and natural cytotoxicity by retinoic acid

The present study has shown that in vitro antibody-dependent cellular cytotoxicity and natural cytotoxicity of human blood lymphocytes are inhibited by retinoic acid (5 X 10(-4)M). The inhibition of cytotoxicity is due to a specific effect on the effector lymphocytes and not to an effect on the target cells. The reduction of killing is most pronounced with prolonged incubation (20 h) of the killer cells with retinoic acid, recovery of killer and natural killer cell activity occurring with post-treatment incubation (greater than 24 h, 37 degrees C) in medium. Retinoic acid was found to have an antagonistic effect to interferon in modulating in vitro natural killer cell activity. The mechanism of inhibition by retinoic acid was uncertain but modulation of cell membrane receptors appeared to be a likely possibility.     

A novel method for retinoic acid administration reveals differential and dose-dependent downregulation of Fgf3 in the developing inner ear and anterior CNS

Endogenous retinoic acid plays critical roles in normal vertebrate development, but can be teratogenic in excess. In mice, additional retinoic acid is administered by oral gavage or intraperitoneal injection. Here we evaluate a novel non-invasive system for administering retinoic acid via chocolate/sugar pellets. We use this delivery system to examine the role of retinoic acid in regulating the expression of the fibroblast growth factor Fgf3, and find that the timing of retinoic acid treatment is critical for its effects on Fgf3 expression. Administration of increasing amounts of retinoic acid at 7.75 dpc leads to dose-dependent downregulation of Fgf3 in the otocyst and changes in spatial expression in the hindbrain. Detailed analysis of the developing inner ear also reveals a lateralisation of Fgf3 expression with increasing retinoic acid dose that is dependent on timing of administration. We discuss how these data impact on current models of retinoic acid patterning of the otocyst.     

Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.     

Inhibition of interferon action by vitamin A.

Simultaneous treatment of mouse cells with interferon and vitamin A (retinoic acid) resulted in an inhibition of interferon action. Increasing concentrations of calf serum decreased the inhibitory effect of retinoic acid on interferon action. Treatment of interferon with retinoic acid prior to the assay for interferon activity also resulted in a loss of interferon activity. Since the residual retinoic acid present after dilution of the interferon for assay was not sufficient to interfere with the assay, it is presumed that interferon and retinoic acid must interact in some fashion to inhibit interferon activity. Calf serum prevented the apparent interaction of retinoic acid and interferon. The loss of interferon activity which resulted from treatment of interferon with retinoic acid was dependent on temperature and time of incubation. Retinyl acetate (acetate ester of vitamin A) and retinal (vitamin A adehyde) only slightly inhibited interferon activity, while retinoic acid (vitamin A acid) and retinol (vitamin A alcohol) were similarly effective at inhibiting interferon activity. Another fat soluble vitamin, vitamin K1, did not inhibit interferon activity.     

Retinoic acid inhibition of cardiac mesenchyme formation in vitro correlates with changes in the secretion of particulate matrix from the myocardium

Retinoic acid has been associated with a variety of cardiac defects. A percentage of these defects are related to changes in the endocardial cushions. Studies in mice and older chick embryos have shown a decrease in mesenchymal cell formation attributable to retinoic acid and have suggested that retinoic acid was affecting the extracellular matrix. In this study we have tested the effect of retinoic acid on cardiac mesenchyme formation in vitro and then tested retinoic acid treated myocyte cultures for changes in the expression of hLAMP-1, fibronectin and transferrin members of the particulate matrix that is required for mesenchyme formation. Initial experiments tested the effect of retinoic acid on mesenchymal cell formation first in atrioventricular canal and outflow tract explant cultures and then in AV endothelial monolayer cultures using myocyte conditioned media or the particulate matrix fraction from retinoic acid treated myocyte cultures. In all cases, mesenchymal cell formation was suppressed while no suppression was observed when MyoCM was included with retinoic acid. Protein analysis showed that retinoic acid had a stimulatory effect on protein synthesis. ELISA assays revealed that retinoic acid treated myocyte cultures contained significantly more hLAMP-1 and fibronectin than either normal or DMSO controls. However, transferrin was not affected by retinoic acid treatment in these experiments. Our results suggest that retinoic acid affects the expression of the particulate matrix and that these changes may be responsible for the observed decrease in mesenchymal cell formation.     

The effect of human recombinant granulocyte-macrophage colony stimulating factor on the proliferation and differentiation of myeloid progenitors in congenital agranulocytosis marrow cells

Congenital agranulocytosis is a rare and frequently fatal infantile disease characterized by recurrent bacterial infections, persistent absence of neutrophils in the peripheral blood and an arrest of myeloid maturation at the promyelocyte/myelocyte stage. The effect of human recombinant Granulocyte-Macrophage colony stimulating factor (GM-CSF) alone and in combination with retinoic acid, dimethylsulphoxide or actinomycin-D on the proliferation and differentiation of bone marrow cells from a child with congenital agranulocytosis was studied. Cells were treated at a concentration of 1 x 10(5) per ml in in-vitro culture with GM-CSF alone and in combination with retinoic acid, dimethylsulphoxide or actinomycin-D for 7 days at 37 degrees C in humidified incubator containing 5% CO2 in air. GM-CSF showed a profound stimulatory effect on the proliferation of myeloid progenitors from the child bone marrow and restored colony numbers in the retinoic acid-, dimethylsulphoxide- and actinomycin-D-inhibited cultures.     

The GABA rho1 subunit interacts with a cellular retinoic acid binding protein in mammalian retina

Interactions between the intracellular domain of ligand-gated membrane receptors and cytoplasmic proteins play important roles in their assembly, clustering, and function. In addition, protein-protein interactions may provide an alternative mechanism by which neurotransmitters activate intracellular pathways. In this study, we report a novel interaction between the GABA rho1 subunit and cellular retinoic acid binding protein in mammalian retina that could serve as a link between the GABA signaling pathway and the control of gene expression in neurons. The interaction between the intracellular loop of the human GABA rho subunit and cellular retinoic acid binding protein was identified using a CytoTrap XR yeast two-hybrid system, and was further confirmed by co-precipitation of the human GABA rho subunit and cellular retinoic acid binding protein from baboon retinal samples. The cellular retinoic acid binding protein binding domain on the human rho1 subunit was located to the C-terminal region of human GABA rho subunit, and the interaction of the human GABA rho subunit with cellular retinoic acid binding protein could be antagonized by a peptide derived from within the binding domain of the rho1 subunit. Since cellular retinoic acid binding protein is a carrier protein for retinoic acid, we investigated the effect of GABA on retinoic acid activity in neuroblastoma cells containing endogenously expressed cellular retinoic acid binding protein. In the absence of the rho1 receptor, these cells showed enhanced neurite outgrowth when exposed to retinoic acid and GABA had no effect on their response to retinoic acid. In contrast, cells stably transfected with the human rho1 subunit showed a significantly reduced sensitivity to retinoic acid when exposed to GABA. These results suggest that the GABA receptor subunit effectively altered gene expression through its interaction with the cellular retinoic acid binding protein pathway.     

Expression of retinoic acid receptor genes in developing rat livers and hepatoma cells.

The expression of retinoic acid receptor alpha, beta, and gamma mRNA was examined in developing rat livers and rat hepatoma-derived cell lines H-4-II-E, McA-RH 7777, and 8994 that represent different hepatocyte phenotypes. Northern blot hybridization demonstrated that all three receptor mRNAs were expressed in the fetal livers of different gestational ages, and the levels of expression increased significantly 3 to 4 weeks after birth. In the hepatoma cell lines, the expression pattern of retinoic acid receptor alpha and gamma mRNA did not correlate with the phenotype. In contrast, retinoic acid receptor beta mRNA was only detected in the adult phenotypic H-4-II-E cells but not in McA-RH 7777 and 8994 cells, which represent embryonic and fetal hepatocyte phenotypes, respectively. The levels of retinoic acid receptor beta mRNA in hepatoma cell lines were lower than adult rat liver. These data suggest that the increased expression of retinoic acid receptor beta gene is associated with differentiation or maturation of rat hepatocytes. The effect of retinoic acid on retinoic acid receptor gene expression was also studied in hepatoma cells. Retinoic acid did not regulate retinoic acid receptor gene expression in McA-RH 7777 and 8994 cells, and the retinoic acid receptor beta gene remained inactivated in these cells. However, Southern blot hybridization indicated that the gross structure of retinoic acid receptor beta gene was not altered during malignant transformation. In H-4-II-E cells, retinoic acid increased the expression of retinoic acid receptor beta and gamma gene. Because of the similarity between H-4-II-E cells and normal adult hepatocytes, this type of autoregulation may be a mechanism by which retinoic acid regulates its own effect in vivo.     

Heparan sulfate proteoglycans: key players in cartilage biology

The extracellular matrix (ECM) plays a fundamental role in skeletal patterning and formation of the vertebrate skeleton. This review focuses on the fundamental roles associated with heparan sulfate (HS) proteoglycans in the ECM during cartilage development, which include regulation of gene expression, presentation of growth factors, establishment of morphogen gradients, and modulation of blood homeostasis. The importance of enzymes involved in biosynthesis and assembly of heparan sulfate is also discussed. Finally, the current evidence for functions of individual HS proteoglycans and biosynthetic enzymes based upon human genetic mutation associations with disease and genetic manipulation in transgenic mice is presented. These findings highlight the important role played by HS proteoglycans, such as perlecan, in cartilage development and skeletal growth.     

视黄酸及其受体在机体免疫中的作用

视黄酸(retinoic acid,RA)又名维甲酸是维生素A的活性衍生物,是小分子质量、脂溶性信号分子,通过与其细胞内的受体α(retinoic acid receptorα,RARα)结合发挥生物学功能,在调节生物各种进程如细胞分化、调亡、胚胎发育、再生和视力发育中发挥重要作用。研究表明在很多疾病如炎症性肠病(IBD)、肿瘤等患者体内视黄酸含量减少,给予视黄酸制剂能有效的抑制疾病的进程。目前视黄酸在机体免疫调节中发挥的作用尚不完全明确,有人认为其通过对Th1/Th2和Th17/Treg平衡的调节而发挥在机体抑制炎症的作用,而在肿瘤方面的作用机制则较为复杂。本文将对视黄酸在机体免疫中发挥的作用做一综述。     

The membranous skeleton: the role of cell condensations in vertebrate skeletogenesis

Summary Elements of the vertebrate skeleton are initiated as cell condensations, collectively termed the membranous skeleton whether cartilages or bones by Grüneberg (1963). Condensations, which were identified as the basic cellular units in a recent model of morphological change in development and evolution (Atchley and Hall 1991) are reviewed in this paper. Condensations are initiated either by increased mitotic activity or by aggregation of cells towards a centre. Prechondrogenic (limb bud) and preosteogenic (scierai ossicle) condensations are discussed and contrasted. Both types of skeletogenic condensations arise following epithelial-mesenchymal interactions; condensations are identified as the first cellular product of such tissue interactions. Molecular characteristics of condensations are discussed, including peanut agglutinin lectin, which is used to visualize prechondrogenic condensations, and hyaluronan, hyaladherins, heparan sulphate proteoglycan, chondroitin sulphate proteoglycan, versican, tenascin, syndecan, N-CAM, alkaline phosphatase, retinoic acid and homeobox-containing genes. The importance for the initiation of chondrogenesis or osteogenesis of upper and lower limits to condensation size and the numbers of cells in a condensation are discussed, as illustrated by in vitro studies and by mutant embryos, including Talpid³ in the chick and Brachypod, Congenital hydrocephalus and Phocomelia in the mouse. Evidence that genes specific to the skeletal type are selectively activated at condensation is discussed, as is a recent model involving TGF- and fibronectin in condensation formation. Condensations emerge as a pivotal stage in initiation of the vertebrate skeleton in embryonic development and in the modification of skeletal morphology during evolution.     

Seasonal changes in the lower jaw skeleton in male Atlantic salmon (Salmo salar L.): remodelling and regression of the kype after spawning

The return of Atlantic salmon (Salmon salar) to their home river for spawning coincides with drastic skeletal alterations in both sexes. Most prominent is the development of a kype (hook) at the tip of the lower jaw in males. Salmon that survive spawning have to cope with the kype throughout their life, unless it disappears after spawning, as was suggested in the early literature. To understand the fate of the kype skeleton, we compared morphological and histological features of kypes from pre-spawned mature anadromous males (grilse) with post-spawned males (kelts). The kype of male grilse is supported by fast-growing skeletal needles that differ from regular dentary bone. In kelts, growth of the kype skeleton has stopped and skeletal needles are resorbed apically by osteoclasts. Simultaneously, and despite the critical physiological condition of the animals, proximal parts of the kype skeleton are remodelled and converted into regular dentary bone. Apical resorption of the skeleton explains reports of a decrease of the kype in kelts. The conversion of basal kype skeleton into regular dentary bone contributes to the elongation of the dentary and probably also to the development of a larger kype in repetitive spawning males.