The retinoid X receptor has a critical role in synthetic rexinoid-induced increase in cellular all-trans-retinoic acid.

Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.

Dual role for microbial short-chain fatty acids in modifying SIV disease trajectory following anti-α4ß7 antibody administration.

BACKGROUND: Human Immunodeficiency Virus (HIV)/Simian Immunodeficiency Virus (SIV) infection is associated with significant gut damage, similar to that observed in patients with inflammatory bowel disease (IBD). This pathology includes loss of epithelial integrity, microbial translocation, dysbiosis, and resultant chronic immune activation. Additionally, the levels of all-trans-retinoic acid (atRA) are dramatically attenuated. Data on the therapeutic use of anti-α4ß7 antibodies has shown promise in patients with ulcerative colitis and Crohn's disease. Recent evidence has suggested that the microbiome and short-chain fatty acid (SCFA) metabolites it generates may be critical for anti-α4ß7 efficacy and maintaining intestinal homeostasis. MATERIALS AND METHODS: To determine whether the microbiome contributes to gut homeostasis after anti-α4ß7 antibody administered to SIV-infected rhesus macaques, faecal SCFA concentrations were determined, 16S rRNA sequencing was performed, plasma viral loads were determined, plasma retinoids were measured longitudinally, and gut retinoid synthesis/response gene expression was quantified. RESULTS: Our results suggest that anti-α4ß7 antibody facilitates the return of retinoid metabolism to baseline levels after SIV infection. Furthermore, faecal SCFAs were shown to be associated with retinoid synthesis gene expression and rebound viral loads after therapy interruption. CONCLUSIONS: Taken together, these data demonstrate the therapeutic advantages of anti-α4ß7 antibody administration during HIV/SIV infection and that the efficacy of anti-α4ß7 antibody may depend on microbiome composition and SCFA generation.

Spatial transcriptomics reveals novel genes during the remodelling of the embryonic human arterial valves.

Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.

Exogenous All-Trans Retinoic Acid Induces Myopia and Alters Scleral Biomechanics in Mice.

Purpose: Ocular all-trans retinoic acid (atRA) levels are influenced by visual cues, and exogenous atRA has been shown to increase eye size in chickens and guinea pigs. However, it is not clear whether atRA induces myopic axial elongation via scleral changes. Here, we test the hypothesis that exogenous atRA will induce myopia and alter scleral biomechanics in the mouse. Methods: Male C57BL/6J mice were trained to voluntarily ingest atRA + vehicle (1% atRA in sugar, 25 mg/kg) (RA: n = 16 animals) or vehicle only (Ctrl: n = 14 animals). Refractive error (RE) and ocular biometry were measured at baseline and after 1 and 2 weeks of daily atRA treatment. Eyes were used in ex vivo assays to measure scleral biomechanics (unconfined compression: n = 18), total scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 23), and specific sGAGs (immunohistochemistry: n = 18). Results: Exogenous atRA caused myopic RE and larger vitreous chamber depth (VCD) to develop by 1 week (RE: -3.7 ± 2.2 diopters [D], P < 0.001; VCD: +20.7 ± 15.1 µm, P < 0.001), becoming more severe by 2 weeks (RE: -5.7 ± 2.2 D, P < 0.001; VCD: +32.3 ± 25.8 µm, P < 0.001). The anterior eye biometry was unaffected. While scleral sGAG content was not measurably affected, scleral biomechanics were significantly altered (tensile stiffness: -30% ± 19.5%, P < 0.001; permeability: +60% ± 95.3%, P < 0.001). Conclusions: In mice, atRA treatment results in an axial myopia phenotype. Eyes developed myopic RE and larger VCD without the anterior eye being affected. The decrease in stiffness and increase in permeability of the sclera are consistent with the form-deprivation myopia phenotype.

Functional characterization of interleukin 4 and retinoic acid signaling crosstalk during alternative macrophage activation.

Retinoic acid possesses potent immunomodulatory properties in various cell types, including macrophages. In this study, we first investigated the effects at the transcriptional and functional levels of exogenous retinoic acid in murine bone marrow-derived macrophages (BMDMs) in the presence or absence of interleukin 4 (IL4), a cytokine with potent anti-inflammatory properties. We examined the effect of IL4 on vitamin A homeostasis in macrophages by quantifying retinoid synthesis and secretion. Our RNAseq data show that exogenous retinoic acid synergizes with IL4 to regulate anti-inflammatory pathways such as oxidative phosphorylation and phagocytosis. Efferocytosis and lysosomal degradation assays validated gene expression changes at the functional level. IL4 treatment altered the expression of several genes involved in vitamin A transport and conversion to retinoic acid. Radiolabeling experiments and mass spectrometry assays revealed that IL4 stimulates retinoic acid production and secretion in a signal transducer and activator of transcription 6 (STAT6)-dependent manner. In summary, our studies highlight the key role of exogenous and endogenous retinoic acid in shaping the anti-inflammatory response of macrophages.

Next-generation retinoid X receptor agonists increase ATRA signaling in organotypic epithelium cultures and have distinct effects on receptor dynamics.

Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 µM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.

Altered Structure and Function of Murine Sclera in Form-Deprivation Myopia.

Purpose: The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. Methods: Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE + choroid + sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes). Results: Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P = 0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. Conclusions: We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

The conversion of ß-carotene to vitamin A in adipocytes drives the anti-obesogenic effects of ß-carotene in mice.

OBJECTIVE: The ß-carotene oxygenase 1 (BCO1) is the enzyme responsible for the cleavage of ß-carotene to retinal, the first intermediate in vitamin A formation. Preclinical studies suggest that BCO1 expression is required for dietary ß-carotene to affect lipid metabolism. The goal of this study was to generate a gene therapy strategy that over-expresses BCO1 in the adipose tissue and utilizes the ß-carotene stored in adipocytes to produce vitamin A and reduce obesity. METHODS: We generated a novel adipose-tissue-specific, adeno-associated vector to over-express BCO1 (AT-AAV-BCO1) in murine adipocytes. We tested this vector using a unique model to achieve ß-carotene accumulation in the adipose tissue, in which Bco1-/- mice were fed ß-carotene. An AT-AAV over-expressing green fluorescent protein was utilized as control. We evaluated the adequate delivery route and optimized cellular and organ specificity, dosage, and exposure of our vectors. We also employed morphometric analyses to evaluate the effect of BCO1 expression in adiposity, as well as HPLC and mass spectrometry to quantify ß-carotene and retinoids in tissues, including retinoic acid. RESULTS: AT-AAV-BCO1 infusions in the adipose tissue of the mice resulted in the production of retinoic acid, a vitamin A metabolite with strong effects on gene regulation. AT-AAV-BCO1 treatment also reduced adipose tissue size and adipocyte area by 35% and 30%, respectively. These effects were sex-specific, highlighting the complexity of vitamin A metabolism in mammals. CONCLUSIONS: The over-expression of BCO1 through delivery of an AT-AAV-BCO1 leads to the conversion of ß-carotene to vitamin A in adipocytes, which subsequently results in reduction of adiposity. These studies highlight for the first time the potential of adipose tissue ß-carotene as a target for BCO1 over-expression in the reduction of obesity.

Altered RBP1 Gene Expression Impacts Epithelial Cell Retinoic Acid, Proliferation, and Microenvironment.

Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA.

Role of cellular retinol-binding protein, type 1 and retinoid homeostasis in the adult mouse heart: A multi-omic approach.

The main active metabolite of Vitamin A, all-trans retinoic acid (RA), is required for proper cellular function and tissue organization. Heart development has a well-defined requirement for RA, but there is limited research on the role of RA in the adult heart. Homeostasis of RA includes regulation of membrane receptors, chaperones, enzymes, and nuclear receptors. Cellular retinol-binding protein, type 1 (CRBP1), encoded by retinol-binding protein, type 1 (Rbp1), regulates RA homeostasis by delivering vitamin A to enzymes for RA synthesis and protecting it from non-specific oxidation. In this work, a multi-omics approach was used to characterize the effect of CRBP1 loss using the Rbp1-/- mouse. Retinoid homeostasis was disrupted in Rbp1-/- mouse heart tissue, as seen by a 33% and 24% decrease in RA levels in the left and right ventricles, respectively, compared to wild-type mice (WT). To further inform on the effect of disrupted RA homeostasis, we conducted high-throughput targeted metabolomics. A total of 222 metabolite and metabolite combinations were analyzed, with 33 having differential abundance between Rbp1-/- and WT hearts. Additionally, we performed global proteome profiling to further characterize the impact of CRBP1 loss in adult mouse hearts. More than 2606 unique proteins were identified, with 340 proteins having differential expression between Rbp1-/- and WT hearts. Pathway analysis performed on metabolomic and proteomic data revealed pathways related to cellular metabolism and cardiac metabolism were the most disrupted in Rbp1-/- mice. Together, these studies characterize the effect of CRBP1 loss and reduced RA in the adult heart.

MRP5 and MRP9 play a concerted role in male reproduction and mitochondrial function.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

Acute Proteomic Changes in Non-human Primate Kidney after Partial-body Radiation with Minimal Bone Marrow Sparing.

ABSTRACT: Near total body exposure to high-dose ionizing radiation results in organ-specific sequelae, including acute radiation syndromes and delayed effects of acute radiation exposure. Among these sequelae are acute kidney injury and chronic kidney injury. Reports that neither oxidative stress nor inflammation are dominant mechanisms defining radiation nephropathy inspired an unbiased, discovery-based proteomic interrogation in order to identify mechanistic pathways of injury. We quantitatively profiled the proteome of kidney from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Kidney was analyzed by liquid chromatography-tandem mass spectrometry. Out of the 3,432 unique proteins that were identified, we found that 265 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 230 proteins showed strong upregulation while 35 proteins showed downregulation. Bioinformatics analysis revealed significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. These data will be useful for a greater understanding of the molecular mechanisms of injury in well-characterized animal models of partial body irradiation with minimal bone marrow sparing. These data may be potentially useful in the future development of medical countermeasures.

Multi-omic Analysis of Non-human Primate Heart after Partial-body Radiation with Minimal Bone Marrow Sparing.

ABSTRACT: High-dose radiation exposure results in hematopoietic and gastrointestinal acute radiation syndromes followed by delayed effects of acute radiation exposure, which encompasses multiple organs, including heart, kidney, and lung. Here we sought to further characterize the natural history of radiation-induced heart injury via determination of differential protein and metabolite expression in the heart. We quantitatively profiled the proteome and metabolome of left and right ventricle from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Global proteome profiling identified more than 2,200 unique proteins, with 220 and 286 in the left and right ventricles, respectively, showing significant responses across at least three time points compared to baseline levels. High-throughput targeted metabolomics analyzed a total of 229 metabolites and metabolite combinations, with 18 and 22 in the left and right ventricles, respectively, showing significant responses compared to baseline levels. Bioinformatic analysis performed on metabolomic and proteomic data revealed pathways related to inflammation, energy metabolism, and myocardial remodeling were dysregulated. Additionally, we observed dysregulation of the retinoid homeostasis pathway, including significant post-radiation decreases in retinoic acid, an active metabolite of vitamin A. Significant differences between left and right ventricles in the pathology of radiation-induced injury were identified. This multi-omic study characterizes the natural history and molecular mechanisms of radiation-induced heart injury in NHP exposed to PBI with minimal bone marrow sparing.

Acute Proteomic Changes in Lung after Radiation: Toward Identifying Initiating Events of Delayed Effects of Acute Radiation Exposure in Non-human Primate after Partial Body Irradiation with Minimal Bone Marrow Sparing.

ABSTRACT: Radiation-induced lung injury is a delayed effect of acute radiation exposure resulting in pulmonary pneumonitis and fibrosis. Molecular mechanisms that lead to radiation-induced lung injury remain incompletely understood. Using a non-human primate model of partial body irradiation with minimal bone marrow sparing, lung was analyzed from animals irradiated with 12 Gy at timepoints every 4 d up to 21 d after irradiation and compared to non-irradiated (sham) controls. Tryptic digests of lung tissues were analyzed by liquid chromatography-tandem mass spectrometry followed by pathway analysis. Out of the 3,101 unique proteins that were identified, we found that 252 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 215 proteins showed strong up-regulation while 37 proteins showed down-regulation. Canonical pathways affected by irradiation, changes in proteins that serve as upstream regulators, and proteins involved in key processes including inflammation, fibrosis, and retinoic acid signaling were identified. The proteomic profiling of lung conducted here represents an untargeted systems biology approach to identify acute molecular events in the non-human primate lung that could potentially be initiating events for radiation-induced lung injury.

Effect of Radiation on the Essential Nutrient Homeostasis and Signaling of Retinoids in a Non-human Primate Model with Minimal Bone Marrow Sparing.

ABSTRACT: High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. An active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the non-human primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

Transcriptomic, proteomic, and metabolomic analyses identify candidate pathways linking maternal cadmium exposure to altered neurodevelopment and behavior.

Cadmium (Cd) is a ubiquitous toxic heavy metal of major public concern. Despite inefficient placental transfer, maternal Cd exposure impairs fetal growth and development. Increasing evidence from animal models and humans suggests maternal Cd exposure negatively impacts neurodevelopment; however, the underlying molecular mechanisms are unclear. To address this, we utilized multiple -omics approaches in a mouse model of maternal Cd exposure to identify pathways altered in the developing brain. Offspring maternally exposed to Cd presented with enlarged brains proportional to body weights at birth and altered behavior at adulthood. RNA-seq in newborn brains identified exposure-associated increases in Hox gene and myelin marker expression and suggested perturbed retinoic acid (RA) signaling. Proteomic analysis showed altered levels of proteins involved in cellular energy pathways, hypoxic response, and RA signaling. Consistent with transcriptomic and proteomic analyses, we identified increased levels of retinoids in maternally-exposed newborn brains. Metabolomic analyses identified metabolites with significantly altered abundance, supportive of changes to cellular energy pathways and hypoxia. Finally, maternal Cd exposure reduced mitochondrial DNA levels in newborn brains. The identification of multiple pathways perturbed in the developing brain provides a basis for future studies determining the mechanistic links between maternal Cd exposure and altered neurodevelopment and behavior.

Cardiac retinoic acid levels decline in heart failure.

Although low circulating levels of the vitamin A metabolite, all-trans retinoic acid (ATRA), are associated with increased risk of cardiovascular events and all-cause mortality, few studies have addressed whether cardiac retinoid levels are altered in the failing heart. Here, we showed that proteomic analyses of human and guinea pig heart failure (HF) were consistent with a decline in resident cardiac ATRA. Quantitation of the retinoids in ventricular myocardium by mass spectrometry revealed 32% and 39% ATRA decreases in guinea pig HF and in patients with idiopathic dilated cardiomyopathy (IDCM), respectively, despite ample reserves of cardiac vitamin A. ATRA (2 mg/kg/d) was sufficient to mitigate cardiac remodeling and prevent functional decline in guinea pig HF. Although cardiac ATRA declined in guinea pig HF and human IDCM, levels of certain retinoid metabolic enzymes diverged. Specifically, high expression of the ATRA-catabolizing enzyme, CYP26A1, in human IDCM could dampen prospects for an ATRA-based therapy. Pertinently, a pan-CYP26 inhibitor, talarozole, blunted the impact of phenylephrine on ATRA decline and hypertrophy in neonatal rat ventricular myocytes. Taken together, we submit that low cardiac ATRA attenuates the expression of critical ATRA-dependent gene programs in HF and that strategies to normalize ATRA metabolism, like CYP26 inhibition, may have therapeutic potential.

Retinoic acid production, regulation and containment through Zic1, Pitx2c and Cyp26c1 control cranial placode specification.

All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In Xenopus, Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here, we have identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as being crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain. Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that pitx2c is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a program of RA containment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain.

Proteomics of Non-human Primate Plasma after Partial-body Radiation with Minimal Bone Marrow Sparing.

High-dose radiation exposure results in organ-specific sequelae that occurs in a time- and dose-dependent manner. The partial body irradiation with minimal bone marrow sparing model was developed to mimic intentional or accidental radiation exposures in humans where bone marrow sparing is likely and permits the concurrent analysis of coincident short- and long-term damage to organ systems. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the plasma proteome of non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing with 6 MV LINAC-derived photons at 0.80 Gy min over a time period of 3 wk. The plasma proteome was analyzed by liquid chromatography-tandem mass spectrometry. A number of trends were identified in the proteomic data including pronounced protein changes as well as protein changes that were consistently upregulated or downregulated at all time points and dose levels interrogated. Pathway and gene ontology analysis were performed; bioinformatic analysis revealed significant pathway and biological process perturbations post high-dose irradiation and shed light on underlying mechanisms of radiation damage. Additionally, proteins were identified that had the greatest potential to serve as biomarkers for radiation exposure.