Perivascular stromal cells (PSCs) are a recently identified cell type that comprises a small percentage of the platelet derived growth factor receptor-β+ cells within the CNS perivascular space. PSCs are activated following injury to the brain or spinal cord, expand in number and contribute to fibrotic scar formation within the injury site. Beyond fibrosis, their high density in the lesion core makes them a potential significant source of signals that act on neural cells adjacent to the lesion site. Our developmental analysis of PSCs, defined by expression of Collagen1a1 in the maturing brain, revealed that PSCs first appear postnatally and may originate from the meninges. PSCs express many of the same markers as meningeal fibroblasts, including expression of the retinoic acid (RA) synthesis proteins Raldh1 and Raldh2. Using a focal brain ischemia injury model to induce PSC activation and expansion, we show a substantial increase in Raldh1+/Raldh2+ PSCs and Raldh1+ activated macrophages in the lesion core. We find that RA levels are significantly elevated in the ischemic hemisphere and induce signaling in astrocytes and neurons in the peri-infarct region. This study highlights a dual role for activated, non-neural cells where PSCs deposit fibrotic ECM proteins and, along with macrophages, act as a potentially important source of RA, a potent signaling molecule that could influence recovery events in a neuroprotective fashion following brain injury.

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.

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 inHoxgene 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.

Lipids represent biologically ubiquitous and highly dynamic molecules in terms of abundance and structural diversity. Whereas the potential for lipids to inform on disease/injury is promising, their unique characteristics make detection and identification of lipids from biological samples analytically demanding. We report the use of ultraperformance convergence chromatography (UPC²), a variant of supercritical fluid chromatography, coupled to high‐resolution, data‐independent tandem mass spectrometry for characterization of total lipid extracts from mouse lung tissue. The UPC² platform resulted in lipid class separation and when combined with orthogonal column chemistries yielded chromatographic separation of intra‐class species based on acyl chain hydrophobicity. Moreover, the combined approach of using UPC² with orthogonal column chemistries, accurate mass measurements, time‐aligned low‐ and high‐collision energy total ion chromatograms, and positive and negative ion mode product ion spectra correlation allowed for confident lipid identification. Of great interest was the identification of differentially expressed ceramides that were elevated 24 h post whole thorax lung irradiation. The identification of lipids that were elevated 24 h post‐irradiation signifies a unique opportunity to investigate early mechanisms of action prior to the onset of clinical symptoms in the whole thorax lung irradiation mouse model.

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. Significance Multidrug Resistance Proteins (MRPs) are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRP5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.

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.