Vitamin A deficiency and inflammation: the pivotal role of secretory cells in the development of atrophic, hyperplastic and metaplastic change in the tracheal epithelium in vivo.

We showed previously that the proliferation of hamster airway secretory cells decreases during vitamin A deficiency (VAD) but later increases when submucosal inflammation develops (Virchows Arch [B] 59:231-242, 1990). This observation has important biological implications since two morphological extremes (atrophy and quiescence versus hyperplasia and hyperproliferation) are reported in the literature for VAD tracheal epithelium in vivo. In the present study, histological slides of tracheal rings from 35-day-old control and VAD hamsters (Virchows Arch [B] 45:197-219, 1984) were reviewed again. Rings from VAD hamsters were selected based on the absence or presence of a florid submucosal inflammation. Quantitative analyses were made on the cartilaginous part of rings from the anterior third of the trachea. When inflammation was absent, a mucociliary pseudostratified epithelium was, for the most part, maintained. The mitotic rate (MR, 6 h colchicine blockade) of secretory cells was markedly reduced (29-fold) but that of basal cells was not changed significantly. Moreover, cell density was not changed by VAD but ciliated cells and secretory cells were decreased and basal cells were increased, proportionally. We call this "minimal morphological change." Thinning (atrophy) of the minimally changed epithelium was associated with focal cell sloughing. Small scattered foci of epidermoid metaplasia (multiple layers of highly keratinized cells which were extremely flat, so that the epithelium was thin and attenuated) were also seen. We call this "atrophic epidermoid metaplasia." When inflammation was present, hyperplastic changes (stratification and epidermoid metaplasia) predominated and cells were in mitosis at all epithelial levels (low, middle, superficial) except in the most superficial (terminally differentiated) squames. The tracheal epithelium was thickened and hypercellular. The cells were piled up at the stratified lesions, and epithelial height, cell density and epithelial MR were significantly increased compared with the non-inflamed VAD epithelium. The effects of VAD and inflammation on cell proliferation were analyzed further by studying 7 h bromodeoxyuridine (BrdU) labelling patterns of cells in VAD tracheal epithelium, with and without submucosal inflammation. In addition, inflammation was induced in "minimally changed epithelium" by mild mechanical injury. The BrdU labelling patterns confirmed that DNA synthesis by secretory cells is reduced markedly by VAD. However, this suppression is overidden by the influx of inflammatory cells (the nature of the stimulus is unknown). The results indicate that the morphological contrasts (atrophy and hyperplasia) seen in the trachea during VAD in vivo are related to extremes in proliferation rates of tracheal secretory cells, regulated by VAD alone (minimal replication) and by inflammation (maximal replication).     

Shift of graft-versus-host-disease target organ tropism by dietary vitamin A

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4β7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4β7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ(+)CD4(+) and low FoxP3(+)CD4(+) frequencies of total donor CD4(+) T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT.     

Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model

Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.     

Retinoic acid reverses the airway hyperresponsiveness but not the parenchymal defect that is associated with vitamin A deficiency

Airway hyperresponsiveness (AHR) is influenced by structural components of the bronchial wall, including the smooth muscle and connective tissue elements and the neuromuscular function. AHR is also influenced by parenchymally derived tethering forces on the bronchial wall, which maintain airway caliber by producing outward radial traction. Our previous work has shown that vitamin A-deficient (VAD) rats exhibit cholinergic hyperresponsiveness and a decrease in the expression and function of the muscarinic-2 receptors (M2R). We hypothesized that if decreases in radial traction from airway or parenchymal structures contributed to the VAD-related increase in AHR, then the radial traction would normalize more slowly than VAD-related alterations in neurotransmitter signaling. Rats remained vitamin A sufficient (VAS) or were rendered VAD and then maintained on the VAD diet in the presence or absence of supplementation with all-trans retinoic acid (RA). VAD was associated with an approximately twofold increase in respiratory resistance and elastance compared with VAS rats. Exposure to RA for 12 days but not 4 days restored resistance and elastance to control (VAS) levels. In VAD rats, AHR was accompanied by decreases in bronchial M2R gene expression and function, which were restored after 12 days of RA supplementation. Subepithelial bronchial elastic fibers were decreased by approximately 50% in VAD rats and were significantly restored by RA. The increase in AHR that is associated with VAD is accompanied by decreases in M2R expression and function that can be restored by RA and a reduction in airway elastic fibers that can be partially restored by RA.     

Effect of vitamin A deficiency upon gonadotropin response to gonadotropin-releasing hormone

There is a monotypic change in basal serum gonadotropin levels following retinol treatment of chronically vitamin A-deficient (VAD) male rats. The present study was undertaken to investigate the hypothesis that the specific increase in serum follicle-stimulating hormone (FSH) represents a change in gonadotrope responsiveness to gonadotropin-releasing hormone (GnRH). To this end, a test dose of GnRH was given to VAD rats pre-, 5 days post-, and 10 days postreplacement of vitamin A (PVA). In VAD rats, basal serum FSH and luteinizing hormone (LH) levels were higher than those of controls. Increased LH/testosterone ratios, both in basal levels and in the secretory response to GnRH, suggested Leydig cell hyporesponsiveness in VAD animals. Both the FSH and LH responses to GnRH were maximal at 1 h, declining thereafter. Although the absolute increments in FSH and LH 1 h after GnRH in VAD rats were greater than in controls, the percent increase in FSH tended to be lower in VAD rats and to increase after vitamin A replacement. The specific enhancement of FSH release PVA became evident only when assessing total secretion of FSH and LH after GnRH. Luteinizing hormone response to GnRH increased PVA, but not significantly, while FSH secretion after GnRH increased both 5 and 10 days PVA, times during which basal FSH levels were also increasing. These changes in FSH secretion could not be attributed either to increases in endogenous GnRH or to changes in testosterone or estradiol levels. Basal serum androgen binding protein levels, elevated in VAD animals, did not respond to the acute increases in FSH after GnRH and remained high PVA, suggesting no acute change in Sertoli cell function. Thus, the PVA increase in FSH secretion unmasks a partial inhibition of the gonadotrope present in the retinol-deficient, retinoic acid-fed male rat.     

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A‐deficient (VAD) quail embryos lack the vitamin A‐active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFβ2 is involved in RA‐regulated cardiovascular development. In VAD embryos TGFβ2 mRNA and protein expression are greatly elevated. The expression of TGFβ receptor II is also elevated in VAD embryos but is normalized by treatment with TGFβ2‐specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFβ2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFβ2 protein to normal quail embryos mimics the excessive TGFβ2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFβ2 has a major role in the retinoic acid‐regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFβ2 signals during vertebrate heart formation.     

Synergistic effect of vitamin D derivatives and retinoids on C2C12 skeletal muscle cells

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.     

Recent insights on the role and regulation of retinoic acid signaling during epicardial development

The vitamin A metabolite, retinoic acid, carries out essential and conserved roles in vertebrate heart development. Retinoic acid signals via retinoic acid receptors (RAR)/retinoid X receptors (RXRs) heterodimers to induce the expression of genes that control cell fate specification, proliferation, and differentiation. Alterations in retinoic acid levels are often associated with congenital heart defects. Therefore, embryonic levels of retinoic acid need to be carefully regulated through the activity of enzymes, binding proteins and transporters involved in vitamin A metabolism. Here, we review evidence of the complex mechanisms that control the fetal uptake and synthesis of retinoic acid from vitamin A precursors. Next, we highlight recent evidence of the role of retinoic acid in orchestrating myocardial compact zone growth and coronary vascular development.     

The temporal requirement for vitamin A in the developing eye: mechanism of action in optic fissure closure and new roles for the vitamin in regulating cell proliferation and adhesion in the embryonic retina

Mammalian eye development requires vitamin A (retinol, ROL). The role of vitamin A at specific times during eye development was studied in rat fetuses made vitamin A deficient (VAD) after embryonic day (E) 10.5 (late VAD). The optic fissure does not close in late VAD embryos, and severe folding and collapse of the retina is observed at E18.5. Pitx2, a gene required for normal optic fissure closure, is dramatically downregulated in the periocular mesenchyme in late VAD embryos, and dissolution of the basal lamina does not occur at the optic fissure margin. The addition of ROL to late VAD embryos by E12.5 restores Pitx2 expression, supports dissolution of the basal lamina, and prevents coloboma, whereas supplementation at E13.5 does not. Surprisingly, ROL given as late as E13.5 completely prevents folding of the retina despite the presence of an open fetal fissure, showing that coloboma and retinal folding represent distinct VAD-dependent defects. Retinal folding due to VAD is preceded by an overall reduction in the percentage of cyclin D1 positive cells in the developing retina, (initially resulting in retinal thinning), as well as a dramatic reduction in the cell adhesion-related molecules, N-cadherin and β-catenin. Reduction of retinal cell number combined with a loss of the normal cell-cell adhesion proteins may contribute to the collapse and folding of the retina that occurs in late VAD fetuses.     

Vitamin A and Feeding Statuses Modulate the Insulin-Regulated Gene Expression in Zucker Lean and Fatty Primary Rat Hepatocytes

Unattended hepatic insulin resistance predisposes individuals to dyslipidemia, type 2 diabetes and many other metabolic complications. The mechanism of hepatic insulin resistance at the gene expression level remains unrevealed. To examine the effects of vitamin A (VA), total energy intake and feeding conditions on the insulin-regulated gene expression in primary hepatocytes of Zucker lean (ZL) and fatty (ZF) rats, we analyze the expression levels of hepatic model genes in response to the treatments of insulin and retinoic acid (RA). We report that the insulin- and RA-regulated glucokinase, sterol regulatory element-binding protein-1c and cytosolic form of phosphoenolpyruvate carboxykinase expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS) diet ad libitum. The impairments are partially corrected when ZF rats are fed a VA deficient (VAD) diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. These results demonstrate that VA and feeding statuses modulate the hepatic insulin sensitivity at the gene expression level.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

PPARgamma ligands suppress proliferation of human urothelial basal cells in vitro

Expression of peroxisome proliferator-activated receptor (PPAR) gamma in the human urinary tract through embryonic development suggests its possible roles in the development, proliferation, and differentiation of uroepithelium. Little is known, however, about physiological roles of PPARgamma in the urinary tract. We investigated effects of PPARgamma ligands on the proliferation of normal human urothelial cells and stromal cells cultivated from surgical specimens. Active proliferation in vitro as well as high molecular weight cytokeratin expression indicated that cultured urothelial cells possess basal cell phenotype. PPARgamma protein, expressed predominantly in the epithelial layer of the normal human urinary tract in vivo, was abundantly expressed in urothelial cells but barely detectable in stromal cells in vitro. Natural ligand for PPARgamma, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), as well as synthetic ones, troglitazone and pioglitazone, suppressed proliferation of the urothelial cells dose-dependently. These effects were PPARgamma specific because clofibrate or PGF(2alpha) did not affect proliferation of urothelial cells. Neither 9-cis retinoic acid or all-trans retinoic acid (ATRA) at 1 microM showed any synergism on the antiproliferative effects of PPARgamma ligands. Urothelial cells treated with PPARgamma ligands showed drastic morphologic changes and cell cycle arrest at G0/G1 phase accompanied with increased mRNA level of a cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Since 15d-PGJ(2) is present in vivo during the resolution phase of inflammation, these results indicated that PPARgamma might be involved in the terminal phase of urothelial re-epithelialization processes.     

Effect of tobacco extract on surfactant synthesis and its reversal by retinoic acid—role of cell–cell interactions in vitro

Tobacco induces oxidative stress in the alveolar epithelium and causes its damage. Retinoic acid (RA) has a cardinal role in alveolar cell growth, differentiation, and maturation. The aim of the study was to investigate the role of cell–cell interactions and whether RA could reverse the effect of tobacco extract on epithelial function as expressed by surfactant synthesis. For this, an in vitro model, which provides multiple cell type interactions, as seen in vivo, was used. We had used the major lung cell types, alveolar epithelial and mesenchymal cells represented by the cell lines A549 (human lung adenocarcinoma cell line), and human fetal lung fibroblast-1 (HFL-1) for developing the monoculture and co-culture systems and studied the effect of tobacco extract and retinoic acid. The effect of tobacco and retinoic acid both singly and in combination on proliferation and surfactant synthesis was analyzed. Retinoic acid induced proliferation and upregulated surfactant synthesis in monocultures and co-cultures. Tobacco extract at 100 μg/ml concentration decreased A549 proliferation and upregulated surfactant protein mRNA expression. In co-cultures treated with tobacco extract (100 μg/ml), retinoic acid (1 μM), regulated cell proliferation, and surfactant protein mRNA expression vis-à-vis the monoculture system. This clearly points to the fact that cell–cell interactions modulate the effect of additives or stimulants and help in assessing the in vivo combinatorial responses in vitro and that the retinoic acid effect is regenerative.     

Vitamin A deficiency alters the pulmonary parenchymal elastic modulus and elastic fiber concentration in rats

Background

Bronchial hyperreactivity is influenced by properties of the conducting airways and the surrounding pulmonary parenchyma, which is tethered to the conducting airways. Vitamin A deficiency (VAD) is associated with an increase in airway hyperreactivity in rats and a decrease in the volume density of alveoli and alveolar ducts. To better define the effects of VAD on the mechanical properties of the pulmonary parenchyma, we have studied the elastic modulus, elastic fibers and elastin gene-expression in rats with VAD, which were supplemented with retinoic acid (RA) or remained unsupplemented.

Methods

Parenchymal mechanics were assessed before and after the administration of carbamylcholine (CCh) by determining the bulk and shear moduli of lungs that that had been removed from rats which were vitamin A deficient or received a control diet. Elastin mRNA and insoluble elastin were quantified and elastic fibers were enumerated using morphometric methods. Additional morphometric studies were performed to assess airway contraction and alveolar distortion.

Results

VAD produced an approximately 2-fold augmentation in the CCh-mediated increase of the bulk modulus and a significant dampening of the increase in shear modulus after CCh, compared to vitamin A sufficient (VAS) rats. RA-supplementation for up to 21 days did not reverse the effects of VAD on the elastic modulus. VAD was also associated with a decrease in the concentration of parenchymal elastic fibers, which was restored and was accompanied by an increase in tropoelastin mRNA after 12 days of RA-treatment. Lung elastin, which was resistant to 0.1 N NaOH at 98°, decreased in VAD and was not restored after 21 days of RA-treatment.

Conclusion

Alterations in parenchymal mechanics and structure contribute to bronchial hyperreactivity in VAD but they are not reversed by RA-treatment, in contrast to the VAD-related alterations in the airways.