Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological ( approximately 1 microM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.     

Growth hormone and insulin-like growth factor I regulate collagen gene expression and extracellular collagen in cultures of avian skin fibroblasts

Avian skin fibroblasts were isolated, cultured and incubated with [³H]proline for 24 h. The cells exported radiolabeled collagenase-digestible (CDP) and non-collagenase-digestible (NCDP) proteins into the medium. Human, bovine and avian growth hormone (GH) as well as insulin-like growth factor I (IGF-I) attenuated the appearance of [³H]CDP in the medium without affecting [³H]NCDP. The appearance of [³H]CDP was not affected by prolactin. The effects of GH and IGF-I were enhanced by increasing concentrations of fetal calf serum (FCS). A synergism was observed between GH and IGF-I in their effect on CDP. Each peptide, at an ineffective concentration, increased the sensitivity of the cells to the other peptide. Collagenase activity in the medium was enhanced by IGF-I, but not modified by GH, FCS, or by their interaction with IGF-I. GH and IGF-I inhibition of type I procollagen gene expression was demonstrated with the aid of probes containing sequences corresponding to the mRNAs for avian I and II chains. The results suggest that GH and IGF-I cooperate in regulating collagen synthesis, but collagen degradation is affected by IGF-I and not by GH.     

Comparison of Endothelin-1 and Noradrenaline Stimulated Inositol Phosphate Formation in Cultured Aortic Smooth Muscle Cells from Spontaneously Hypertensive and Wistar Kyoto Rats

Total [³H]-inositol phosphate formation was measured in cultured aortic smooth muscle cells from 6 and 14 week spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) normotensive rats. Basal inositol phosphate formation was significantly increased in cells cultured from SHR compared to WKY at both 6 and 14 weeks as was basal phosphatidylinositol formation. This difference in basal values was apparent after 9 h or more incubation with [³H]-myoinositol. Both endothelin-1 and noradrenaline stimulated inositol phosphate formation was unchanged in cultured smooth muscle cells from 6-week SHR compared to WKY. In cultured smooth muscle cells from 14-week SHR a decrease was observed in endothelin-1 stimulated inositol phosphate formation compared to controls. Noradrenaline stimulated inositol phosphate formation was increased in cultured cells from 14 week SHR. Endothelin-1 and noradrenaline stimulated inositol phosphate formation does not appear to be involved in the development (at 6 weeks) of hypertension in this model. However, in established hypertension (14 weeks) cells from SHR have altered total [³H] inositol phosphate formation in response to stimulation with noradrenaline and endothelin-1 although these changes are in opposite directions. Therefore, in cultured smooth muscle cells from 14-week rats noradrenaline and endothelin-1 appear to be regulated independently with regard to their effects on the phosphatidylinositol cycle.     

Plasma levels of retinoids, carotenoids and tocopherols in patients with mild obstructive sleep apnoea

Background and objective:   OSA is associated with increased incidence of cardiovascular diseases. Pathogenic mechanisms of vascular diseases include thickened vascular walls due to the increased number of smooth muscle cells (SMC). Retinoic acid (RA) suppresses the growth of SMC, and reduced retinoid levels are associated with vascular diseases. Oxidant signalling promotes SMC growth, thus antioxidant levels may also influence the development of cardiovascular diseases. The present study tested the hypothesis that plasmas from OSA patients contain altered levels of retinoids, carotenoids and tocopherols.
Methods:   Plasma samples were taken before and after sleep from patients with OSA (mostly mild) without known cardiovascular diseases and from control subjects. Levels of retinoids, carotenoids and tocopherols were measured using sensitive gas chromatograph-mass spectrometry and high pressure liquid chromatography methods and total antioxidant capacity was assessed fluorometrically.
Results:   Results showed that plasmas from patients with OSA had significantly lower retinyl palmitate and 9- cis RA compared with control subjects, while levels of retinol, all- trans RA and 13- cis RA were indifferent. All- trans β-carotene and 9- cis β-carotene were also lower in OSA patients. Levels of all- trans RA and 13- cis RA in OSA patients were reduced after sleep compared with before sleep. OSA patients showed significantly higher δ-tocopherol compared with controls. Treatment of cultured human vascular SMC with post-sleep OSA patient plasmas promoted cell growth, but not in controls.
Conclusions:   Mild OSA exhibits altered levels of specific retinoids, carotenoids and tocopherols, which may be markers and/or mediators for the increased susceptibility of patients to vascular diseases.     

Desensitization of LLC-PK1 cells by vasopressin results in receptor down-regulation

The molecular mechanism of desensitization of antidiuretic hormone receptors is not well understood. Preincubation of LLC-PK₁ cells with lysine vasopressin (LVP) (10⁻⁶ M, 5 h) decreased subsequent LVP-stimulated cAMP accumulation in cells by 83% and reduced the V_max of LVP-stimulated adenylate cyclase by 81%. Such preincubation also reduced by 90% the binding of [³H]LVP to both intact cells and isolated plasma membranes, suggesting a loss of vasopressin receptors. Both the reduction in cAMP response and the apparent loss of receptors showed similar dose and time dependence. Monensin (33 μM) did not alter [³H]LVP binding or stimulation of cAMP by LVP, nor did it prevent desensitization. However, membranes prepared from cells preincubated with LVP in the presence of monensin did not show a decrease in [³H]LVP binding. Forskolin preincubation, at 0.1, 1, 10 and 100 μM, did not alter [³H]LVP binding or accumulation of cellular cAMP by LVP, nor did it induce desensitization to LVP. Cells desensitized with varying LVP concentrations in the presence of 10 μM forskolin displayed the same loss of [³H]LVP binding and LVP responsiveness as observed in the absence of forskolin. LVP-desensitized cells, upon removal from LVP-containing medium, recovered cAMP responsiveness to LVP and specific binding of [³H]LVP at the same rate, achieving control levels after 50 h. Recovery was prevented by cycloheximide (25 μg/ml). These findings are consistent with a desensitization process involving LVPmediated receptor internalization, and a recovery process requiring protein synthesis.     

Effects of retinoids on iodine metabolism, thyroid peroxidase gene expression, and deoxyribonucleic acid synthesis in porcine thyroid cells in culture.

Effects of retinoids on DNA synthesis, iodine metabolism, and thyroid peroxidase messenger RNA levels were studied in cultured porcine thyroid cells. Retinol (10(-8)-10(-5) M) alone did not affect DNA synthesis but potentiated that induced by epidermal growth factor or insulin-like growth factor-I without changes in the number or affinity of receptors for the growth factors, suggesting that retinol stimulates postreceptor events responsible for DNA synthesis. Retinol was an inhibitor of TSH-stimulated iodine metabolism. Iodide uptake and release of organified iodine stimulated by TSH or forskolin were inhibited dose dependently by treatment with retinol. The inhibition was detected at 10(-8) M and was approximately 50% at 10(-6) M. The potency of retinoic acid was comparable to that of retinol. The inhibitory effect of retinol was detected after treatments of thyroid cells for 24 h, and the maximal effect occurred after 48 h incubation. The cAMP accumulation in cultures treated with TSH plus retinol was lower than that of control cultures treated with TSH alone. However, iodide uptake stimulated by 8-bromo-cAMP was also inhibited by retinoids. Retinol reduced TSH- or 8-bromo-cAMP-stimulated gene expression of thyroid peroxidase. Thus, the data suggest that retinoids inhibit TSH-stimulated iodine metabolism by reducing cAMP accumulation and also by acting on the steps subsequent to cAMP production.     

Pulmonary surfactant transport in alveolar type II cells

Abstract:   Pulmonary surfactant (PS) is a mixture of several lipids (mainly phosphatidylcholine; PC) and four apoproteins (A, B, C and D). The classical hypothesis of PS transport suggests that PS is synthesized in the endoplasmic reticulum and transported to the lamellar body (LB) via the Golgi apparatus. However, recent studies have raised questions regarding this single route. This study examined, independently, the intracellular trafficking route of three different components of PS, that is, PC, SP-A and SP-B. Alveolar type II cells were isolated from Sprague–Dawley rats or Japanese white rabbits. The cells were cultured with either [³H]choline or [³⁵S]methionine/cysteine with or without brefeldin A, which disassembles the Golgi apparatus. LB was purified from disintegrated cells with sucrose density gradient centrifugation. [³H]PC was extracted from radiolabeled media, cells, and the LB fraction with Bligh–Dyer's method. [³⁵S]SP-A or [³⁵S]SP-B was immunoprecipitated from each sample with a specific antibody. [³H]PC was transported and stored to the LB via a Golgi-independent pathway. [³⁵S]SP-A was transported to the Golgi apparatus, underwent glycosylation, and was then constitutively secreted. The secreted [³⁵S]SP-A was re-uptaken into the LB. [³⁵S]SP-B was transported and stored to the LB via the Golgi-dependent pathway. These results indicate that, rather than a single route, surfactant components take different pathways to reside in the LB. These different pathways may reflect the different nature and role of each surfactant component such as surface tension-lowering activity and innate host defense.     

STAT3-dependent mouse embryonic stem cell differentiation into cardiomyocytes: analysis of molecular signaling and therapeutic efficacy of cardiomyocyte precommitted mES transplantation in a mouse model of myocardial infarction

Pluripotent embryonic stem (ES) cell therapy may be an attractive source for postinfarction myocardial repair and regeneration. However, the specific stimuli and signal pathways that may control ES cell-mediated cardiomyogenesis remains to be completely defined. The aim of the present study was to investigate (1) the effect and underlying signal transduction pathways of leukemia inhibitory factor (LIF) and bone-morphogenic protein-2 (BMP-2)-induced mouse ES cell (mES-D3 line) differentiation into cardiomyocytes (CMC) and (2) the efficacy of CMC precommitted mES cells for functional and anatomical cardiac repair in surgically-induced mouse acute myocardial infarction (AMI) model. Various doses of LIF and BMP-2 and their inhibitors or blocking antibodies were tested for mES differentiation to CMC in vitro. CMC differentiation was assessed by mRNA and protein expression of CMC-specific markers, Connexin-43, CTI, CTT, Mef2c, Tbx5, Nkx2.5, GATA-4, and alphaMHC. LIF and BMP-2 synergistically induced the expression of CMC markers as early as 2 to 4 days in culture. Signaling studies identified STAT3 and MAP kinase (ERK1/2) as specific signaling components of LIF+BMP-2-mediated CMC differentiation. Inhibition of either STAT3 or MAPK activation by specific inhibitors drastically suppressed LIF+BMP-2-mediated CMC differentiation. Moreover, in mouse AMI, transplantation of lentivirus-GFP-transduced, LIF+BMP-2 precommitted mES cells, improved post-MI left ventricular functions, and enhanced capillary density. Transplanted cells engrafted in myocardium and differentiated into CMC and endothelial cells. Our data suggest that LIF and BMP-2 may synergistically enhance CMC differentiation of transplanted stem cells. Thus augmentation of LIF/BMP-2 downstream signaling components or cell type specific precommitment may facilitate the effects of ES cell-based therapies for post-MI myocardial repair and regeneration.     

Lack of difference between retinoic acid and retinol in stimulating progesterone production by luteinizing granulosa cells in vitro

Receptors for retinoids in the immature rat ovary and the effects of retinol and retinoic acid on luteinizing granulosa cells were studied. Radioreceptor assay demonstrated the presence of specific cellular retinol-binding protein and cellular retinoic acid-binding protein in the ovaries of rats injected with PMSG alone or PMSG and hCG. In addition, when luteinizing granulosa cell from PMSG/hCG-injected immature rats were cultured with or without retinoic acid, the morphology, viability, number of cells in culture, and progesterone (P) accumulation were not affected by up to 10 microM retinoic acid. Beyond 10 microM, the cells began to round up, which was associated with a decrease in cell viability. Surprisingly, the deleterious concentrations of retinoic acid increased progesterone accumulation significantly higher than the medium control value. This increase in progesterone, however, was not accompanied by an increase in cAMP. When cells preincubated for 2 days with 1 microM of either retinoic acid or retinol were subsequently incubated in retinoid-free medium containing various substrates for steroidogenesis, the following results were obtained. Basal progesterone and its accumulation in response to human low density lipoprotein were significantly higher in cells preincubated with retinoids than in the control cells. However, no difference was seen in the degree of stimulation between retinol and retinoic acid pretreatments. Both 25-hydroxycholesterol, a substrate for side-chain cleavage enzyme, and pregnenolone, a substrate for 3 beta-hydroxysteroid dehydrogenase, significantly stimulated the accumulation of progesterone in cells preincubated with retinoids over the control value. Again, no appreciable difference was observed between retinol and retinoic acid pretreatments. Our results suggest that receptors for retinoids are present in gonadotropin-primed immature rat ovaries, retinoids increase luteal cell progesterone accumulation, and no difference exists between retinol and retinoic acid in their ability to increase the accumulation of progesterone by these cells.     

The levels of leukemia inhibitory factor in synovial tissues of patients with rheumatoid arthritis: inflammation and other proinflammatory cytokines

 To clarify the effect of leukemia inhibitory factor (LIF) on the destruction of rheumatoid arthritis (RA) joints, we investigated the production of LIF and the expression of LIF mRNA in synovial tissues from patients with RA and osteoarthritis (OA). Synovial fluids from RA were used to measure the LIF concentrations using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistory and RT-PCR were used to examine the expression of LIF by synovial cells. LIF mRNA was detected in all cases in RA synovial cells. Although LIF protein was detected only in 20 cases (19%) in RA synovial fluids, LIF concentration in the synovial fluids significantly correlated with the peripheral leukocyte count (P < 0.001) and C-reactive protein (CRP) (P < 0.01). Moreover, levels of IL-1β, IL-6, and IL-8, but not TNF-α, were significantly correlated with LIF in the RA synovial fluids. LIF production was promoted by IL-1β and TNF-α stimulation; in contrast, IL-1 ra and IL-4 were found to markedly decrease LIF production by cultured synovial cells. LIF appeared to be a cytokine produced by RA synovium leading to a proinflammatory secretion profile. Moreover, IL-4 and IL-1 ra may represent attenuated activity for reducing the effect of the destruction of joints by LIF. Received: February 12, 2002 / Accepted: September 6, 2002     

Age-differences in choline acetyltransferase activities and muscarinic receptor binding in brain regions of C57BL/6J mice

Muscarinic receptor binding and choline acetyltransferase (ChAT, EC.2.3.1.6.) activity were assayed in four brain regions of C57BL/6J mice of four ages (4, 12, 18, and 24 months). In the cerebellum, there were no age differences in either of the cholinergic markers. However, significant age differences were noted in the V_max for ChAT and in the B_max for [³H]quinuclidinyl benzilate ([³H]QNB) in the cortex, striatum and hippocampus. Increases were noted in V_max of the synthetic enzyme in all three regions and for B_max in the hippocampus. B_max for [³H]QNB decreased in the cortex and striatum. The high- and lower-affinity muscarinic binding constants, the percentage of muscarinic binding to high affinity sites determined by carbamylcholine displacement of [³H]QNB, as well as the affinities of ChAT for acetyl coenzyme A and choline chloride showed no age differences in any brain region.     

An autoradiographic study of [(18)F]FDG uptake to islets of Langerhans in NOD mouse

To evaluate the potential of in vivo imaging of accumulation of lymphocytes to islets of Langerhans (insulitis), we compared 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) uptake in the pancreas and pancreatic islets of healthy BALB/c mice, phenotypically healthy NOD mice with insulitis and diabetic NOD mice. [¹⁸F]FDG was injected i.v. to 14 female BALB/c mice (age 13 ± 3 weeks, plasma glucose 8 ± 2 mmol/l) and 21 age-matched female NOD mice (plasma glucose 8 ± 4 mmol/l, p = 0.06). The mice were killed 90-min post injection and distribution of radioactivity was analysed using digital autoradiography. There was no correlation of plasma glucose concentration with the [¹⁸F]FDG uptake values. Uptake of radioactivity in NOD mice to the islets affected by insulitis was up to 2.3 times higher (p = 0.001) than that to unaffected islets in the same pancreas. Uptake to NOD islets with insulitis was also clearly enhanced (1.0–2.3 times higher) compared to the islets in the BALB/c mice.

In conclusion, NOD mouse islets with insulitis accumulate [¹⁸F]FDG markedly more than islets without insulitis or BALB/c islets. However, the relatively small difference in the [¹⁸F]FDG intensity between healthy and diseased islets, combined with the limited resolution ability of the positron emission tomography (PET), probably prevent the use of [¹⁸F]FDG in PET studies aiming at in vivo documentation of onset and progression of insulitis and prediabetes in mouse and man.     

不同病因肝衰竭患者外周血单个核细胞HLA-DR基因水平及血Th17和CD4+CD25+Treg细胞百分比的变化

目的探讨不同病因所致肝衰竭患者外周血单个核细胞（PBMCs）HLA-DR mRNA及Th17和CD4⁺CD25⁺Treg细胞水平的变化及其意义。方法本研究纳入肝衰竭患者50例,其中乙型肝炎肝衰竭15例,药物性肝损伤12例,酒精性肝病13例,自身免疫性肝炎10例;慢性乙型肝炎患者17例和正常人10例。采用PCR法检测PBMCs中HLA-DR mRNA水平,使用流式细胞仪检测CD4⁺CD25⁺Treg和Th17细胞百分比。结果乙型肝炎肝衰竭患者HLA-DR mRNA水平为（134.5±15.2）,显著高于药物性肝损伤组的（17.9±1.2）、酒精性肝病组的（19.6±2.0）和自身免疫性肝炎组的[（11.2±1.2）,P<0.05];不同病因肝衰竭患者Th17和CD4⁺CD25⁺Treg细胞百分比[分别为（4.4±0.6）%和（3.9±0.6）%左右]的差异无统计学意义（P>0.05）,但与慢性乙型肝炎[分别为（3.7±0.2）%和（6.1±0.4）%和正常人（2.1±0.7）%和（7.0±0.9）%比,均有显著性差异（P<0.05）;对不同病因肝衰竭患者进行动态观察发现,19例死亡患者CD4⁺CD25⁺Treg细胞百分比呈持续下降,直至死亡,而31例生存患者则逐渐恢复至接近正常水平。结论外周血单个核细胞HLA-DR mRNA水平及Th17和CD4⁺CD25⁺Treg细胞百分比的变化与肝衰竭患者的病情密切相关,可作为判断肝衰竭严重程度及预后的指标。     

The levels of leukemia inhibitory factor in synovial tissues of patients with rheumatoid arthritis: inflammation and other proinflammatory cytokines

Abstract

To clarify the effect of leukemia inhibitory factor (LIF) on the destruction of rheumatoid arthritis (RA) joints, we investigated the production of LIF and the expression of LIF mRNA in synovial tissues from patients with RA and osteoarthritis (OA). Synovial fluids from RA were used to measure the LIF concentrations using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistory and RT-PCR were used to examine the expression of LIF by synovial cells. LIF mRNA was detected in all cases in RA synovial cells. Although LIF protein was detected only in 20 cases (19%) in RA synovial fluids, LIF concentration in the synovial fluids significantly correlated with the peripheral leukocyte count (P < 0.001) and C-reactive protein (CRP) (P < 0.01). Moreover, levels of IL-1β, IL-6, and IL-8, but not TNF-α, were significantly correlated with LIF in the RA synovial fluids. LIF production was promoted by IL-1β and TNF-α stimulation; in contrast, IL-1 ra and IL-4 were found to markedly decrease LIF production by cultured synovial cells. LIF appeared to be a cytokine produced by RA synovium leading to a proinflammatory secretion profile. Moreover, IL-4 and IL-1 ra may represent attenuated activity for reducing the effect of the destruction of joints by LIF.     

Insulin-like growth factor-I and -II in the ovary of a bony fish, Oreochromis mossambicus, the tilapia: in situ hybridisation, immunohistochemical localisation, Northern blot and cDNA sequences

In FRTL-5 cells, cultured over a period of more than 3 years, different properties of the cells have been observed to undergo spontaneous changes in the course of aging, i.e. after an increase in the number of passages. This consists mainly in alterations in their morphological phenotype and in some of their functional properties. The morphology of the cells displayed a progressive disruption of the monolayer organization with a loss of cell–cell contacts and a marked rounding-up of the cells. The uptake of iodide was not modified nor was the expression of thyroglobulin (Tg) mRNA as determined at various time intervals in the course of the cells culturing. Estimation of the proliferation by counting the frequency of [³H]thymidine labeled nuclei revealed an age-related decline in the sensitivity to TSH mitogenic action associated with a reciprocal increase in the insulin synergistic effect. Aged cells (±40 passages) lost their apoptosis sensitivity to the phosphatase inhibitor, okadaic acid (OA) but not to cycloheximide (CHX) and/or actinomycin D (act. D) exposure. Altogether these observations favor the existence of a shift towards transformed properties with only partial loss of differentiated functions.     

Expression of leukemia inhibitory factor (LIF) and its receptor gp190 in human liver and in cultured human liver myofibroblasts. Cloning of new isoforms of LIF mRNA

BACKGROUND: The cytokine leukemia inhibitory factor (LIF) mediates its biological effects through binding to its high affinity receptor made of the low-affinity LIF receptor subunit gp190 (LIF-R) and the gp130 subunit. LIF exerts several important effects in the liver, however, data on liver expression of LIF are scarce. The aim of this study was to examine the expression of LIF and LIF-R in human liver. RESULTS: LIF expression, analyzed by immunohistochemistry, was barely detectable in normal liver but was strong within cirrhotic fibrous septa and was found in spindle-shaped cells compatible with myofibroblasts. Accordingly, cultured human liver myofibroblasts expressed high levels of LIF as shown by ELISA and Northern blot. Biological assay demonstrated that myofibroblast-derived LIF was fully active. RT-PCR showed expression of the LIF-D and M isoforms, and also of low levels of new variants of LIF-D and LIF-M resulting from deletion of exon 2 through alternative splicing. LIF receptor expression was detected mainly as a continuous sinusoidal staining that was enhanced in cirrhotic liver, suggestive of endothelial cell and/or hepatocyte labeling. Immunohistochemistry, flow cytometry and STAT-3 phosphorylation assays did not provide evidence for LIF receptor expression by myofibroblasts themselves. LIF secretion by cultured myofibroblasts was down regulated by the addition of interleukin-4. CONCLUSIONS: We show for the first time the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of LIF mRNA. Expression of LIF by myofibroblasts and of its receptor by adjacent cells suggests a potential LIF paracrine loop in human liver that may play a role in the regulation of intra-hepatic inflammation.     

Rat hepatic stellate cells become retinoid unresponsive during activation.

Hepatic stellate cells (HSC) play an essential role in fibrogenesis. Many stimuli cause HSC to activate, lose their Vitamin A and produce collagen. It is unclear whether Vitamin A loss causes activation, potentiates it or is simply an event in the cascade of activation changes. We determine if exogenous retinoids prevent the activation of freshly isolated rat HSC activated by plating on plastic. We also determine if retinoids: (1) reverse HSC activation; (2) maintain/restore HSC intracellular retinoid levels; (3) maintain expression of HSC nuclear receptors for retinoic acid (RAR) in HSC that are becoming activated or are chronically activated. Markers of activation in freshly isolated HSC were decreased by either retinol or retinoic acid without increases in HSC retinoid concentration. mRNA levels for RAR-alpha, RAR-beta and RAR-gamma, the nuclear receptors for retinoic acid, decreased during activation of freshly isolated HSC even with retinoid supplementation. RAR-alpha, RAR-beta and RAR-gamma mRNA and RAR-beta protein was undetectable in chronically activated HSC and remained absent after retinoic acid supplementation. Activation markers in chronically activated HSC were only slightly decreased after retinoid exposure. We conclude that exposure of HSC to extracellular retinoids diminishes some markers of activation but does not prevent HSC activation.     

Influence of retinoids on growth and melanin content of Harding-Passey-melanoma cells in vitro and B16 transplantable melanoma in vivo

Growth cessation and cell death of exponentially proliferating Harding-Passey melanoma cells (HPM-73 line) in monolayer culture resulted in the presence of 3.3 X 10(-5) M retinal, while retinol and retinoic acid caused growth retardation at 3.3 X 10(-5) M. Also at 1 X 10(-5) M, the growth-inhibitory effect of retinal was more pronounced than that of retinol or retinoic acid. Following serum removal from the culture media, all 3 retinoids at 3.3 X 10(-6) M or 1 X 10(-5) M revealed cytotoxic effects within 3 days as demonstrated by cell loss from the substratum. Thus, the presence of serum has "protective" effects. Addition of retinal, retinoic acid or retinol at 1 X 10(-5) M to cultures in stationary growth phase did not result in cell loss during period of 6 days. C57Bl mice with B16 melanotic melanoma were i.p. injected during 10 days with retinoids (30 or 100 mcg per mouse daily). All retinoids inhibited B16 tumor growth in vivo. In this respect, retinoic acid was the most effective one. The cellular melanin content of cultured HPM-cells and of B16 melanotic melanoma in vivo was elevated after treatment with retinoids; retinal having the strongest effect.