Stimulation by retinoic acid of prostaglandin production and its inhibition by tumor promoters in mouse myeloid leukemia cells

Retinoic acid induced lysozyme activity in mouse myeloid leukemia M1 cells. It also stimulated the synthesis and release of prostaglandins such as prostaglandin F2alpha, E2, and D2 by the cells. The particulate fraction of retinoic acid-treated M1 cells converted arachidonate to prostaglandins, and this conversion was almost completely inhibited by indomethacin. Retinol, retinal and retinyl acetate, but not the pyridyl analog of retinoic acid, also induced lysozyme activity and stimulated synthesis and release of prostaglandins. Indomethacin inhibited the induction of lysozyme activity by retinoic acid. The induction of lysozyme activity and the stimulation of prostaglandin E2 production were dependent on the concentration of retinoic acid. Kinetic studies showed that stimulation of prostaglandin E2 production by retinoic acid was followed by induction of lysozyme activity. The tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol 12,13-didecanoate inhibited the induction of lysozyme activity by retinoic acid, but 4 alpha-phorbol didecanoate and phorbol did not. TPA and phorbol 12, 13-didecanoate, but not 4 alpha -phorbol didecanoate, also inhibited the stimulation of prostaglandin E2 production by retinoic acid. These results suggest that stimulation by retinoic acid of prostaglandin E2 production in M1 cells is a prerequisite for the induction of lysozyme activity. On the other hand, both retinoic acid and TPA inhibited the induction by dexamethasone of phagocytic activity, which is a typical functional marker of differentiation of M1 cells, without causing significant growth inhibition. Suboptimal concentrations of retinoic acid and TPA had synergistic inhibitory effects on the induction of phagocytic activity of M1 cells by dexamethasone.     

Regulation of neurotrophin receptor expression by retinoic acid in mouse sympathetic neuroblasts

We have studied the effect of retinoic acid on the expression of the neurotrophin receptors trkA, trkC, and p75 by neuroblasts and neurons at different axial levels along the embryonic mouse paravertebral sympathetic chain. In dissociated cultures of sympathetic neuroblasts, retinoic acid inhibited the developmental increase in trkA mRNA expression and the developmental decrease in trkC mRNA expression that normally occurs in these cells but did not affect p75 mRNA expression. At higher concentrations, retinoic acid also increased the proliferation of sympathetic neuroblasts. After sympathetic neuroblasts became postmitotic, retinoic acid no longer affected receptor expression. Studies with retinoic acid receptor agonists and antagonists indicated that the effects of retinoic acid on neurotrophin receptor expression were mediated mainly by alpha retinoic acid receptors, not beta or gamma receptors. The observation that alpha-antagonists increased trkA mRNA expression in intact sympathetic ganglion explants suggests that endogenous retinoic acid is a physiological regulator of trkA receptor expression.     

Antisense retinoic acid receptor gamma-1 oligonucleotide enhances chondrogenesis of mouse limb mesenchymal cells in vitro

Retinoic acid receptor (RAR) gamma gene is expressed in the precartilaginous cells during chondrogenesis in mouse embryos, but the role of the gene products is still unclear. To examine the role during chondrogenesis, we isolated mesenchymal cells from the limb bud of mouse embryos and exposed them to antisense RAR gamma-1 oligodeoxynucleotide in micromass culture. The antisense oligodeoxynucleotide inhibited RAR gamma-1 protein expression and enhanced chondrogenesis in the exposed cells. These results suggest that the complex of RAR gamma-1 protein and its ligand RA acts as a suppressor of the chondrogenesis in the limb development.     

Inhibition of cholesteatoma migration in vitro with all-trans retinoic acid

Retinoids have recently become of interest to clinicians because of their ability to inhibit migration and proliferation of premalignant squamous cells while enhancing growth and proliferation of normal cells. An in vitro investigation was undertaken to determine whether retinoic acid exhibits similar inhibitory effects on cholesteatoma cells. Cholesteatoma specimens were obtained intraoperatively from 10 patients undergoing mastoidectomy or revision mastoidectomy for chronic ear disease. Cholesteatoma explant growth and en mass migration were observed daily, and topographic maps were constructed at various time intervals to quantity rate and direction of explant migration in the presence or absence of all-trans retinoic acid. Before all-trans retinoic acid administration, explants migrated very rapidly (1 to 2 mm/day). A maximum threefold inhibition of migratory rate occurred, with explants exposed to 0.1 micromol/L retinoic acid when compared with controls. A sixfold maximum inhibition was observed at higher retinoic acid concentrations (5 micromol/L). On removal of all-trans retinoic acid, twofold and fourfold increases in migratory rates were observed. The direction of explant migration varied significantly for long periods of time and appeared not to be affected by retinoic acid. This investigation suggests that all-trans retinoic acid has an inhibitory effect on cholesteatoma cell migration. Retinoids may have a role in controlling cholesteatoma disease in the future.     

Retinoic acid receptor-gamma in human epidermis preferentially traps all-trans retinoic acid as its ligand rather than 9-cis retinoic acid

Brain injury in the premature infant is an extremely important problem, in part because of the large absolute number of infants affected yearly. The 2 principal brain lesions that underlie the neurological manifestations subsequently observed in premature infants are periventricular hemorrhagic infarction and periventricular leukomalacia. The emphases of this article are the neuropathological features, pathogenesis, and potential means of prevention of these 2 lesions. Recent work suggests that the ultimate goal, prevention of the lesions, is potentially achievable. Periventricular hemorrhagic infarction may be avoidable by prevention of germinal matrix-intraventricular hemorrhage, and periventricular leukomalacia by detection of impaired cerebrovascular autoregulation, prevention of impaired cerebral blood flow, and interruption of the cascade to oligodendroglial cell death by such agents as free radical scavengers.     

Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus

We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha). The sequence is highly homologous in all RARs and their isoforms. When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal. Mouse RAR-gamma gave an additional signal corresponding to 58 K. In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected. The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment. The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary. Using immunohistochemistry the RARs were found in the nuclear compartment. In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells. In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells.     

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all-trans RA into 5,8-epoxy all-trans RA. P450RA metabolizes other biologically active RAs such as 9-cis RA and 13-cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all-trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage- and region-specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.     

In vivo and in vitro association of retinoic acid with form-deprivation myopia in the chick

A relationship between survival and high cardiac index and oxygen delivery (DO2) has been documented for patients of all ages. Debate continues whether achieving these parameters decreases mortality or whether these parameters merely reflect better physiologic reserve. Younger patients who spontaneously generate high DO2 have low mortality rates and do not present a treatment challenge. The difficulty and controversy concern the use of inotropic agents and blood transfusions in older patients with concurrent myocardial dysfunction who are unable to mount an appropriate DO2 response to increased oxygen demands. Although it is obvious that one DO2 value may not satisfy all patients, there are difficulties in recognizing areas of tissue ischemia, and practioners attempt to keep DO2 > or = 600 mL/min/m2 to ensure tissue perfusion. Logically, assessment of oxygen needs in the elderly should be based on patients' sex, age, muscle mass, premorbid activity level as well as the disease state. Although DO2 augmentation to > or = 600 mL/min/m2 may be appropriate for most critically ill patients, a 450 to 550 mL/min/m2 value may be equivalent to a "high" DO2 in relation to the basal needs for the very old (age > 75 yrs). Keeping oxygen extraction ratios < or = 0.25 during early resuscitation may be used as an additional guide in titrating DO2. Technology which allows identification of ischemic areas may assist in guiding individual treatment rather than utilizing a global DO2, goal.     

Effects of retinoic acid on lipoprotein lipase activity and mRNA level in vitro and in vivo

This study was designed to determine whether all-trans retinoic acid altered lipoprotein lipase (LPL) activity and mRNA levels in vitro and tissue LPL mRNA levels in vivo. Incubation of adipocytes or adipose tissue for up to 12 hr with 10(-6) or 10(-5) M all-trans retinoic acid did not decrease LPL activity. There was no change in LPL mRNA levels following 3 hr incubation of adipocytes with all-trans retinoic acid. Feeding all-trans retinoic acid for 4 days led to a significant decrease in adipose tissue LPL activity but no change in heart enzyme activity. Retinoic acid did not alter the increase in heart LPL activity observed with fasting. There were no changes in LPL mRNA levels in adipose tissue, heart or liver. Retinoic acid does not have an acute direct effect on adipose tissue LPL activity. The observed decrease in adipose tissue LPL activity in vivo is not due to alterations in mRNA levels and may be a secondary effect of retinoic acid.     

Spatial distributions of retinoic acid receptor gene transcripts in the prenatal mouse inner ear

Thirty-one patients with a presentation compatible with an acquired demyelinating polyradiculoneuropathy were prospectively evaluated for proximal conduction block using root stimulation (RS) of cervical and/or lumbar roots. These same techniques were applied to 78 controls (40 pathological). Proximal conduction block was noted in 20/31 cases and none of the controls. Only 7 of 31 cases met published demyelinating criteria. Intravenous immune globulin therapy was completed by 21/31 patients and 20/21 responded. RS is superior to published electrophysiologic criteria for identifying demyelinating polyneuropathies and predicting who will respond to treatment. RS should be performed in all patients suspected of having an acquired demyelinating polyneuropathy when traditional nerve conduction studies criteria are nondiagnostic. RS reliably predicts those likely to respond to immunosuppressive therapy.     

Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos

The concentration/effect relationship of bunazosin, a selective alpha-1-adrenoceptor antagonist of the quinazoline class, was established in hypertensive patients after a first dose of bunazosin and after 8 weeks of chronic dosing. The study was part of a placebo controlled, multicenter dose-finding trial with parallel group design. After a wash-out period and a 2-week placebo run-in phase, each 4 patients received either placebo, 3 mg, 6 mg or 12 mg bunazosin o.d. in a slow-release formulation. Concentration/effect analysis based on a sigmoidal Emax-model and the drop in the mean arterial blood pressure application of this model was feasible in 9/11 patients. The EC50 after the first dose was 4.6 +/_ 2.0 ng/ml and increased to 9.1 +/- 7.3 ng/ml at steady-state. The decrease in the average of the responsiveness parameter was mainly due to about three-fold, right-shift in the concentration/effect curves at steady-state in 3 patients, the remaining patients showed no or considerably lesser changes of the EC50. Whereas a significant correlation was observed between the pretreatment mean arterial blood pressure and the height of the maximal effect under bunazosin, no relationship appears between the bunazosin dose, the resulting plasma levels at trough and the corresponding effect.     

Genetic control of the development by retinoic acid

Two families of nuclear receptors for retinoic acid (RA) have been characterized. Members of the RAR family (types alpha, beta and gamma and their isoforms alpha 1, alpha 2, beta 1 to beta 4, and gamma 1 and gamma 2) are activated by most physiologically occurring retinoids (all-trans RA, 9-cis RA, 4oxo RA and 3,4 dihyroRA). In contrast, members of the RXR family (types alpha, beta and gamma and their isoforms) are activated by 9cis-RA only. In addition to the multiplicity of receptors, the complexity of retinoid signalling is further increased by the fact that, at least in vitro, RARs bind to their cognate response elements as heterodimers with RXRs. Moreover, RXRs can also bind, in vitro, to some DNA elements as homodimers, and are heterodimeric partners for other nuclear receptors, including TRs, VDR, PPARs and a number of orphan nuclear receptors. To evaluate the functions of the different RARs and RXRs types and isoforms, we have generated null mutant mice by targeted gene disruption in ES cells. As to the functions of RARs, we found that RAR alpha 1 and RAR gamma 2 null mutant mice are apparently normal. Mice deficient in RAR alpha or RAR gamma (i.e., all alpha or gamma isoforms disrupted) show aspects of the post-natal vitamin A deficiency (VAD) syndrome which can be cured or prevented by RA, including post-natal lethality, poor weight gain and male sterility. RAR beta 2 (and RAR beta) null mutants display a retrolenticular membrane which represents the most frequent defect of the fetal VAD syndrome. That these abnormalities were restricted to a small subset of the tissues normally expressing these receptors suggested that some degree of functional redundancy should exist in the RAR family. To test this hypothesis we then generated RAR double null mutants. RAR alpha beta, RAR alpha gamma and RAR beta gamma compound mutants exhibit all the malformations of the fetal VAD syndrome, thus demonstrating that RA is the vitamin A derivative which plays a crucial role at many different stages and in different structures during organogenesis. Interestingly, almost all the structures derived from mesenchymal neural crests cells (NCC) are affected in RAR compound mutants. As to the functions of RXRs, RXR gamma null mutants are viable, fertile and morphologically normal. In contrast, RXR alpha null fetuses display a thin ventricular wall and die in utero from cardiac failure. A myocardial hypoplasia has also been observed in some RAR compound mutants as well as in VAD fetuses. Thus, RXR alpha seems to act as an inhibitor of ventricular cardiocyte differentiation and/or as a positive regulator of their proliferation, and these functions might involve heterodimerization with RARs and activation by RA. RXR beta null mutants are viable but the males are sterile, most probably because of an abnormal lipid metabolism in the Sertoli cells. New abnormalities, absent in RXR alpha mutants, are generated in RXR alpha/RAR (alpha, beta or gamma) compound mutants. All these abnormalities are also seen in RAR double mutants as well as in VAD fetuses. In contrast, such manifestations of synergism are not observed between the RXR beta or RXR gamma and the RAR (alpha, beta or gamma) null mutations. These data strongly support the conclusion that RXR alpha/RAR heterodimers represent the main functional units of the RA signalling pathway during embryonic development. Moreover, since RXR gamma-/-/RXR beta-/-/RXR alpha +/-mutants are viable, a single allele of RXR alpha can perform most of the developmental RXR functions.     

Altered wound arginine metabolism by corticosterone and retinoic acid

The objective of this study is to study the pharmacokinetics of imipenem-cilastatin in patients with anuria related to multiorgan failure during continuous venovenous hemodialysis (CVVHD) at a fixed blood flow rate of 60 ml/min, fixed dialysate flow rate of 20 ml/min, and drainage flow rate of 1-3 mil/min. A prospective open label study was designed in an intensive care unit in a university hospital. Six patients who required mechanical ventilation and inotropic agents and exhibited acute anuric renal failure were examined. Intravenous imipenem-cilastatin, 500/500 mg, was administered over 30 mins. Blood samples were obtained from the inlet and the outlet of the dialyzer and dialysate samples from the outlet before and at 0.0, 0.5, 1.0, 2.0, 3.0, 6.0, 9.0, and 12.0 hrs after infusion ended. The peak and trough concentrations of imipenem were 32.47 +/- 6.69 micrograms/ml and 1.12 +/- 0.46 micrograms/ml, respectively, whereas those of cilastatin were 50.05 micrograms/ml +/- 14.80 and 9.53 +/- 3.25 micrograms/ml, respectively. The half life was 2.79 +/- 0.3 hr for imipenem, and 6.67 +/- 0.93 hr for cilastatin. Total body clearance of imipenem was 89.4 +/- 17.5 ml/min, including the clearance by CVVHD of 18.7 +/- 1.2 ml/min, and corresponding values for cilastatin were 31.7 +/- 9.0 ml/min and 13.7 +/- 3.4 ml/min. In conclusion, in patients with anuria, the elimination of imipenem-cilastatin was constant during CVVHD. The recommended regimen in patients with anuria who receive CVVHD in this setting was estimated as intravenous imipenem-cilastatin, 500/500 mg, every 12 hrs.     

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.     

Regulation of IRF and STAT gene expression by retinoic acid

Ex vivo production of cytokines as determined by whole blood stimulation and supernatant ELISA is partly determined by heritability. To assess the ability of this system to distinguish between high and low producers the laboratory error and individual variation were investigated. Whole blood samples from healthy volunteers were collected using endotoxin-free tubes and were incubated with 0 to 1000 ng/ml lipopolysaccharide concentrations for 4 and 24 h, and subsequently centrifuged. In the supernatants, TNF-alpha and IL10 were measured by ELISA. Coefficients of variation for the day-to-day variation in the blood sampling, transport and stimulation as well as in the whole blood stimulation per se ranged from 7.5% to 12.3%. The intra-individual variation was 15% (TNF-alpha) and 19% (IL10) in contrast to the inter-individual variation of, on average, 35%. No interchanging of ranks between high and low producers was observed after repeating the whole blood stimulation on distinct days. The whole blood stimulation system is able to distinguish high and low producers of TNF-alpha and IL10.     

Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid

The antiphospholipid antibodies (aPL) present in "antiphospholipid-protein syndrome and autoimmune disorders" are associated with thromboembolic episodes, such as venous and/or arterial thrombosis and fetal loss. Patients with antiphospholipid antibodies have, by definition, laboratory abnormalities in either coagulation assays or various solid phase immunoassays ELISA or radioimmunoassays (RIA). These assay systems were initially thought to detect antibodies against phospholipids. The problem was complicated when it was reported that phospholipid is not the sole antigen but only a part of it, the other contribution being due to b2-glycoprotein I (b2-GP I). More findings, demonstrate that the aPL are in fact anti-b2-GP I antibodies directed against a epitope which is expressed when b2-GP I is bound to anionic phospholipid or another suitable surface. Recent studies have demonstrated that antibodies related to lupus anticoagulant (LA) induce an anticoagulant activity in b2-GP I. Some of these LA require binding to phospholipid. However, not all LA require b2-GP I as a cofactor. Human prothrombin is an antigen for some LA IgG's. Finally, a subclassification of phospholipid-dependent coagulation test anticoagulants is described, there appear to be several subclasses of LA, and the clinical and laboratory criteria required to establish the diagnosis of antiphospholipid-protein syndrome is emphasised.     

Cell motility is inhibited by the antiepileptic compound, valproic acid and its teratogenic analogues

Valproic acid (VPA) is an established human teratogen that causes neural tube defects in 1-2% of human foetuses exposed to the drug during early pregnancy. In this study, individual cell motility was evaluated using short- and long-term time-lapse video-recording and computer assisted image analysis, and it was found that VPA and selected VPA-analogues inhibited individual cell motility of L-cells in a dose-dependent manner. The compounds caused a decrease in the root-mean-square speed, S, and in the rate of diffusion, R, but an increase in the time of persistence in direction, P. Using short-term recordings and measurements of mean-cell speed, the reduction in the motile behaviour was shown to correlate with the teratogenic potency of the tested compounds. The observed effects of VPA on cell motility was independent of the employed L-cell clone, and could be reproduced in cells containing the neuronal marker NCAM and in the neuronal cell line N2a. Furthermore, the observed effect was independent of culture substratum, being observed for L-cells grown on fibronectin as well as on plastic. Immunofluorescence microscopy revealed that VPA-treatment of mouse L-cells caused a redistribution of F-actin and of a series of focal adhesion proteins, indicating that the effect of VPA on cell motility may be causally related to increased cell-substratum interactions or to alterations in the organisation or dynamics of the actin cytoskeleton.     

Enhancement of fractionated-dose irradiation by retinoic acid plus interferon

PURPOSE: To evaluate the relative cytotoxicity of fractionated-dose radiation in the presence and absence of 13-cis-retinoic acid (RA) plus alpha-2a-interferon (IFN), as a function of overall treatment time. METHODS AND MATERIALS: Studies were performed with the human squamous cell carcinoma line FaDu, in vitro. Attached exponential phase cells were treated with RA + IFN for 8-10 h and then exposed to single graded doses of radiation, or 1 to 6 doses of radiation at 2 Gy per dose, or to 5 doses of radiation at 2 Gy/dose with a time interval of 4-24 h between treatments. Following irradiation, the cells were incubated with drugs present throughout colony formation, and the fraction of survivors in the presence and absence of the combined drugs was calculated. RESULTS: For single graded-dose irradiation, the surviving fraction ratio at 2 Gy in the absence vs. presence of drugs was 1.27 +/- 0.19 in 3 repeat experiments. Following administration of 6 doses of radiation at 2 Gy/fraction with a 5-h time interval between treatments and, after correcting for cell proliferation between treatments, the surviving fractions differed by a factor of 3.25, again indicating an average difference in survival of 1.26 after each of the 6 2-Gy/fractions. Treatment with 5 2-Gy doses of irradiation with 24 vs. 4 h elapsing between doses, resulted in a 3-fold greater decrease in survival in the presence of drugs vs. no drug. The relatively greater cell kill due to 24 vs. 4 h between treatments was due to drug inhibition of cell proliferation between the more prolonged treatments. CONCLUSIONS: The results of this study indicate that retinoic acid plus interferon both sensitizes and inhibits cell proliferation during treatment. These results suggest that this combination of radiation and drugs, when used concurrently, may be effective for inhibiting tumor cell proliferation or accelerated repopulation during clinical fractionated radiotherapy.