Correction of Defective T-Regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

Recent advances in the design of RAR α and RAR ß agonists as orally bioavailable drugs. A review.

Retinoic acid receptors (RARs) α, ß, and γ are members of the nuclear receptor superfamily. Compounds which bind to and activate the RARs are termed retinoids which regulate a wide variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, and apoptosis, as well as their disorders. Although many synthetic selective RARα, RARß, and RARγ agonists have been designed and prepared, these have generally been lipophilic acids without good drug-like properties and with low oral bioavailability. Recently this has been changing and drug design approaches to highly potent and selective RARα and RARß agonists with low lipophilicity that are orally bioavailable and less toxic have been developed, that have a range of potential therapeutic uses. This review covers these new advances.

All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

2-Bromopalmitate targets retinoic acid receptor alpha and overcomes all-trans retinoic acid resistance of acute promyelocytic leukemia.

Fatty acid oxidation dependency of leukemia cells has been documented in recent studies. Pharmacologic inhibition of fatty acid oxidation, thereby, displays significant effects in suppressing leukemia. 2-Bromopalmitate, a palmitate analogue, was initially identified as an inhibitor of fatty acid oxidation, and recently recognized as an inhibitor of protein palmitoylation. However, the effects of 2-Bromopalmitate on leukemia and its cellular targets remain obscure. Herein, we discover in cultured cell lines, a transplantable mouse model, and primary blasts that 2-Bromopalmitate presents synergistic differentiation induction with all-trans retinoic acid in acute promyelocytic leukemia. Moreover, 2-Bromopalmitate overcomes all-trans retinoic acid resistance in all-trans retinoic acid-resistant cells and leukemic mice. Mechanistically, 2-Bromopalmitate covalently binds at cysteine 105 and cysteine 174 of retinoic acid receptor alpha (RARα) and stabilizes RARα protein in the presence of all-trans retinoic acid which is known to induce RARα degradation, leading to enhanced transcription of RARα-target genes. Mutation of both cysteines largely abrogates the synergistic effect of 2-Bromopalmitate on all-trans retinoic acid-induced differentiation, demonstrating that 2-Bromopalmitate promotes all-trans retinoic acid-induced differentiation through binding RARα. All-trans retinoic acid-based regimens including arsenic trioxide or chemotherapy, as preferred therapy for acute promyelocytic leukemia, induce adverse events and irreversible resistance. We expect that combining all-trans retinoic acid with 2-Bromopalmitate would be a promising therapeutic strategy for acute promyelocytic leukemia, especially for overcoming all-trans retinoic acid resistance of relapsed acute promyelocytic leukemia patients.

In Silico Identification and Experimental Validation of (-)-Muqubilin A, a Marine Norterpene Peroxide, as PPARα/γ-RXRα Agonist and RARα Positive Allosteric Modulator.

The nuclear receptors (NRs) RARα, RXRα, PPARα, and PPARγ represent promising pharmacological targets for the treatment of neurodegenerative diseases. In the search for molecules able to simultaneously target all the above-mentioned NRs, we screened an in-house developed molecular database using a ligand-based approach, identifying (-)-Muqubilin (Muq), a cyclic peroxide norterpene from a marine sponge, as a potential hit. The ability of this compound to stably and effectively bind these NRs was assessed by molecular docking and molecular dynamics simulations. Muq recapitulated all the main interactions of a canonical full agonist for RXRα and both PPARα and PPARγ, whereas the binding mode toward RARα showed peculiar features potentially impairing its activity as full agonist. Luciferase assays confirmed that Muq acts as a full agonist for RXRα, PPARα, and PPARγ with an activity in the low- to sub-micromolar range. On the other hand, in the case of RAR, a very weak agonist activity was observed in the micromolar range. Quite surprisingly, we found that Muq is a positive allosteric modulator for RARα, as both luciferase assays and in vivo analysis using a zebrafish transgenic retinoic acid (RA) reporter line showed that co-administration of Muq with RA produced a potent synergistic enhancement of RARα activation and RA signaling.

Design and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist.

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARß, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARß (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Retinoic acid induction of CD1d expression primes chronic lymphocytic leukemia B cells for killing by CD8+ invariant natural killer T cells.

Invariant natural killer T (iNKT) cells are cytotoxic T cells that respond to glycolipid antigens presented by CD1d. Therapeutic activation of iNKT cells with α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in mice and clinical trials involving α-GalCer-stimulated iNKT cells are ongoing in humans. B cells express CD1d, however, we show that CD1d expression is reduced on B cells from patients with chronic lymphocytic leukemia (CLL). B cells from CLL patients pulsed with α-GalCer failed to stimulate cytolytic degranulation by iNKT cell lines, but could present the more potent glycolipid analogue, 7DW8-5. Retinoic acid receptor-α (RAR-α) agonists induced CD1d expression by CLL B cells, restoring their ability to present α-GalCer to CD8α+ iNKT cells, resulting in cytolytic degranulation. Thus, RAR-α agonists can augment the anti-tumor activities of iNKT cells against CLL cells in vitro. Their inclusion in iNKT cell-based therapies may benefit patients with CLL.

Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action.

Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARß/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARß) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to >60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARß compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.

RARα/RXR synergism potentiates retinoid responsiveness in cutaneous T-cell lymphoma cell lines.

Retinoids, natural and synthetic derivatives of vitamin A, induce cellular changes by activating nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Although the ability of retinoids to govern gene expression is exploited clinically for cancer therapeutics, the full benefit of retinoid-based strategies is unrealized due to detrimental side effects. Delineating the receptors that prompt cellular outcomes is critical to advancing retinoid-based approaches. Here, we identify the receptors that evoke multiple responses in cutaneous T-cell lymphoma (CTCL). The data demonstrate that RARα drives integrin ß7-dependent adhesion and CCR9-mediated chemotaxis in CTCL cells. Of note, concomitant activation of RARα and RXR nuclear receptors yielded synergistic increases in adhesion and migration at concentrations where single agents were ineffective. As the established paradigm of retinoid action in CTCL is apoptosis and growth arrest, the role of RARα/RXR in these events was studied. As with adhesion and migration, RARα/RXR synergism prompted apoptosis and dampened CTCL cell proliferation. Strikingly, RARα/RXR synergism induced responses from CTCL cell lines previously reported to be unresponsive to retinoids. These data provide a novel framework that may further refine a proven CTCL therapy.

Radiolabeling and initial biological evaluation of [18F]KBM-1 for imaging RAR-α receptors in neuroblastoma.

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [18F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [18F]KBM-1 was carried out through KHF2 assisted substitution of [18F]- from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [18F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5min to 60min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30min to 60min post injection. Tumor uptake in subset of 30min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [18F]KBM-1 as a RAR-α imaging agent.

Design, Synthesis and Evaluation of Indene Derivatives as Retinoic Acid Receptor α Agonists.

A series of novel indene-derived retinoic acid receptor α (RARα) agonists have been designed and synthesized. The use of receptor binding, cell proliferation and cell differentiation assays demonstrated that most of these compounds exhibited moderate RARα binding activity and potent antiproliferative activity. In particular, 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)-carbamoyl)benzoic acid (36d), which showed a moderate binding affinity, exhibited a great potential to induce the differentiation of NB4 cells (68.88% at 5 µM). Importantly, our work established indene as a promising skeleton for the development of novel RARα agonists.

Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity.

Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH)2D3, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

The activation of RARα prevents surgery-induced cognitive impairments via the inhibition of neuroinflammation and the restoration of synaptic proteins in elderly mice.

Post-operative cognitive dysfunction (POCD) is a multi-etiological symptom mainly occurred in elderly people after surgery. The activation of retinoic acid receptor α (RARα), a transcriptional factor, was previously predicated to be negatively associated with the occurrence of POCD. However, the mechanisms underlying anti-POCD effects of RARα were still unclear. In this study, AM580, a selective agonist of RARα, and all-trans-retinoic acid (ATRA), a pan agonist of RAR, significantly alleviated cognitive dysfunction and increased the expression of RARα in elderly mice after surgery, which was decreased by RO41-5253, an antagonist of RARα. A bioinformatic study further predicted that the activation of RARα might produce anti-POCD effects via the restoration of synaptic proteins. Both agonists inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (Myd88) and the phosphorylation of nuclear factorkappa-B (NF-κB), leading to the prevention of microglial over-activation and pro-inflammatory cytokines secretion in the hippocampal regions of elderly mice after surgery. Moreover, AM580 and ATRA increased the expression of brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95), and the phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element binding protein (CREB). All these results suggested that the activation of RARα prevented surgery-induced cognitive impairments via the inhibition of neuroinflammation by the reduction of the TLR4/Myd88/NF-κB pathway and the restoration of synaptic proteins by the activation of the BDNF/ERK/CREB pathway, providing a further support that RARα could be developed as a therapeutic target for POCD.

Inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells by a retinoic acid receptor-α agonist.

Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-ß2 (TGF-ß2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-ß2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-ß2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-ß2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

Convergence of distinct signaling pathways on synaptic scaling to trigger rapid antidepressant action.

Ketamine is a noncompetitive glutamatergic N-methyl-d-aspartate receptor (NMDAR) antagonist that exerts rapid antidepressant effects. Preclinical studies identify eukaryotic elongation factor 2 kinase (eEF2K) signaling as essential for the rapid antidepressant action of ketamine. Here, we combine genetic, electrophysiological, and pharmacological strategies to investigate the role of eEF2K in synaptic function and find that acute, but not chronic, inhibition of eEF2K activity induces rapid synaptic scaling in the hippocampus. Retinoic acid (RA) signaling also elicits a similar form of rapid synaptic scaling in the hippocampus, which we observe is independent of eEF2K functioni. The RA signaling pathway is not required for ketamine-mediated antidepressant action; however, direct activation of the retinoic acid receptor α (RARα) evokes rapid antidepressant action resembling ketamine. Our findings show that ketamine and RARα activation independently elicit a similar form of multiplicative synaptic scaling that is causal for rapid antidepressant action.

Hemodynamics-Based Strategy of Using Retinoic Acid Receptor and Retinoid X Receptor Agonists to Induce MicroRNA-10a and Inhibit Atherosclerotic Lesion.

The protocols in this chapter describe methods for identifying the functional roles of retinoic acid receptor (RAR) and retinoid X receptor (RXR) signaling in atherosclerosis and developing RARα/RXRα-specific agonists as hemodynamics-based therapeutic components for atherosclerosis treatment. In vitro cell culture flow system is used to elucidate the effects of different flow patterns and shear stresses, i.e., atherogenic oscillatory shear stress (OS) vs. atheroprotective pulsatile shear stress (PS), on RAR/RXR signaling and inflammatory responses in vascular endothelial cells (ECs). Western blotting, nuclear and cytoplasmic protein extraction, immunoprecipitation, and in situ proximity ligation assay are used to examine the expression, location, and association of RARs (i.e., RARα, RARß, and RARγ) and RXRs (i.e., RXRα, RXRß, and RXRγ) in ECs in response to OS vs. PS. Chromatin immunoprecipitation is used to examine the binding activity of RARα/RA-responsive elements (RARE). RT-microRNA (miR) quantitative real-time PCR and RT-PCR are used to detect the expressions of miR-10a and pro-inflammatory molecules, respectively. Specific siRNAs of RARα and RXRα, precursor miR-10a (PreR-10a), and antagomiR-10a (AMR-10a) are used to elucidate the regulatory roles of RARα, RXRα, and miR-10a in pro-inflammatory signaling in ECs. RARα/RXRα-specific agonists are used to induce miR-10a expression and inhibit OS-induced pro-inflammatory signaling in ECs in vitro. Apolipoprotein E-deficient (ApoE-/-) mice are used as an atherosclerotic animal model. Administration of ApoE-/- mice with RARα/RXRα-specific agonists results in inhibitions in atherosclerotic lesion formation. Co-administration of ApoE-/- mice with RARα/RXRα agonists and AMR-10a is performed to identify the role of miR-10a in RARα/RXRα agonists-mediated inhibition in atherosclerotic lesions. Oil Red O staining and H&E staining are used to examine the levels of atherosclerotic lesions in the vessel wall. In situ miR hybridization and immunohistochemical staining are used to detect the expression of miR-10a and pro-inflammatory molecules and the infiltration of inflammatory cells in the vessel wall. RARα/RXRα-specific agonists are used to mimic the atheroprotective effects of PS to induce endothelial miR-10a and hence repress OS-induced pro-inflammatory signaling and atherosclerotic lesion formation in vivo. The results indicate that RAR/RXR-specific agonists have great potential to be developed as hemodynamics-based therapeutic components for atherosclerosis treatment.

Ro41-5253, a selective antagonist of retinoic acid receptor α, ameliorates chronic unpredictable mild stress-induced depressive-like behaviors in rats: Involvement of regulating HPA axis and improving hippocampal neuronal deficits.

Accumulating reports have highlighted an association between excess retinoids and depression development. Retinoic acid receptor α (RARα) is implicated in the activation of hypothalamus-pituitary-adrenal (HPA) axis and closely involved in the etiology of depression, suggesting it might be a novel target of antidepressant. This study investigated the antidepressant potential of Ro41-5253 (a selective RARα antagonist) and related mechanisms using a depression rat model imitated by social isolation and chronic unpredicted mild stress (CUMS). Sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST) were applied to assess the antidepressant-like effect. HPA axis activity, RARα expression in hypothalamic paraventricular nucleus (PVN) and hypothalamus, and protein levels of brain-derived neurotrophic factor (BDNF) and synapse-related proteins (PSD95, SYP, MAP2) in hippocampus were measured, respectively. We found that Ro41-5253 treatment ameliorated the depressive-like behaviors in CUMS rats, as evidenced by increased sucrose preference in SPT, raised numbers of crossing and rearing in OFT, reduced immobility time and prolonged swimming time in FST. The HPA axis hyperactivity was attenuated by Ro41-5253 (1 mg/kg) treatment, indicated by reduced serum corticosterone level, decreased adrenal gland index, reduced corticotrophin-releasing hormone protein level in hypothalamus, and recovered hypothalamic glucocorticoid receptor protein level. In addition, Ro41-5253 (1 mg/kg) treatment downregulated RARα protein expression in hypothalamic PVN and hypothalamus, and increased the protein levels of BDNF, PSD95, SYP and MAP2 in the hippocampus. We concluded that Ro41-5253 had antidepressant-like effects on CUMS rats by downregulating HPA axis hyperactivity and improving the hippocampal neuronal deficits.

Activation of RARα, RARγ, or RXRα Increases Barrier Tightness in Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells.

The blood-brain barrier (BBB) is critical to central nervous system (CNS) health. Brain microvascular endothelial cells (BMECs) are often used as in vitro BBB models for studying BBB dysfunction and therapeutic screening applications. Human pluripotent stem cells (hPSCs) can be differentiated to cells having key BMEC barrier and transporter properties, offering a renewable, scalable source of human BMECs. hPSC-derived BMECs have previously been shown to respond to all-trans retinoic acid (RA), and the goal of this study was to identify the stages at which differentiating human induced pluripotent stem cells (iPSCs) respond to activation of RA receptors (RARs) to impart BBB phenotypes. Here the authors identified that RA application to iPSC-derived BMECs at days 6-8 of differentiation led to a substantial elevation in transendothelial electrical resistance and induction of VE-cadherin expression. Specific RAR agonists identified RARα, RARγ, and RXRα as receptors capable of inducing barrier phenotypes. Moreover, RAR/RXRα costimulation elevated VE-cadherin expression and improved barrier fidelity to levels that recapitulated the effects of RA. This study elucidates the roles of RA signaling in iPSC-derived BMEC differentiation, and identifies directed agonist approaches that can improve BMEC fidelity for drug screening studies while also distinguishing potential nuclear receptor targets to explore in BBB dysfunction and therapy.