Characterization of Atherosclerotic Mice Reveals a Sex-Dependent Susceptibility to Plaque Calcification but No Major Changes in the Lymphatics in the Arterial Wall.

Lymphatics participate in reverse cholesterol transport, and their presence in the arterial wall of the great vessels and prior experimental results suggest their possible role in the development of atherosclerosis. The aim of this study was to characterize the lymphatic vasculature of the arterial wall in atherosclerosis. Tissue sections and tissue-cleared aortas of wild-type mice unveiled significant differences in the density of the arterial lymphatic network throughout the arterial tree. Male and female Ldlr-/- and ApoE-/- mice on a Western diet showed sex-dependent differences in plaque formation and calcification. Female mice on a Western diet developed more calcification of atherosclerotic plaques than males. The lymphatic vessels within the aortic wall of these mice showed no major changes regarding the number of lymphatic junctions and end points or the lymphatic area. However, female mice on a Western diet showed moderate dilation of lymphatic vessels in the abdominal aorta and exhibited indications of increased peripheral lymphatic function, findings that require further studies to understand the role of lymphatics in the arterial wall during the development of atherosclerosis.

The glycine receptor alpha 3 subunit mRNA expression shows sex-dependent differences in the adult mouse brain.

BACKGROUND: The glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice. RESULTS: Single-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas suggested from the single-cell data. CONCLUSIONS: Glra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.

Identification and characterization of sex-dependent gene expression profile in glioblastoma.

Glioblastoma (GBM) is the most lethal primary tumor in the human brain and lacks favorable treatment options. Sex differences in the outcome of GBM are broadly acknowledged, but the underlying molecular mechanisms remain largely unknown. To identify the sex-dependent critical genes in the progression of GBM, raw data from several microarray datasets with the same array platform were downloaded from the Gene Expression Omnibus (GEO) database. These datasets included tumorous and normal tissue from patients with GBM and crucial sex features. Then, the differentially expressed genes (DEGs) in female and male tumors were identified via bioinformatics analysis, respectively. Functional signatures of the identified DEGs were further annotated by Gene Ontology (GO) and pathway enrichment analyses. Venn diagram and functional protein-protein interaction (PPI) network analyses were performed to screen out the sex-specific DEGs. Survival analysis of patients with differences in the expression level of selected genes was then carried out using the data from The Cancer Genome Atlas (TCGA). Here, we showed that ECT2, AURKA, TYMS, CDK1, NCAPH, CENPU, OIP5, KIF14, ASPM, FBXO5, SGOL2, CASC5, SHCBP1, FN1, LOX, IGFBP3, CSPG4, and CD44 were enriched in female tumor samples, whereas TNFSF13B, CXCL10, CXCL8, CXCR4, TLR2, CCL2, and FCGR2A were enriched in male tumor samples. Among these key genes, interestingly, ECT2 was associated with increased an survival rate for female patients, whileTNFSF13B could be regarded as a potential marker of poor prognosis in male patients. These results suggested that sex differences in patients may be attributed to the heterogeneous gene activity, which might influence the oncogenesis and the outcomes of GBM.

Influences of Taurine Pharmacodynamics and Sex on Active Avoidance Learning and Memory.

Researchers have begun to direct their research to focus on the use of taurine as a psychopharmacotherapeutic compound to treat a wide range of health- related conditions as well as neuropathological diseases. Moreover, taurine has been shown to improve emotional and cognitive declines associated with senescence in neurotypical animal models. However, despite these advances in the field of taurine therapeutics, much less is known regarding the effects of sex and taurine on neurotypical animal models that are then manipulated, modified, and/or mutated to study human diseases. The present study sought to investigate this matter in a Long Evans Hooded rat model of mature age (i.e., postnatal day 60-90) in an active avoidance test (AAT). Rats were trained for 20 trials, given a 1 h. test break, retrained for another 20 trials, and then tested at 24 h, 48 h, and 1 week for learning and memory retention. An N = 63 rats were randomly assigned to three groups: (1) Control (n = 22), (2) Taurine Pre-Train (n = 19), and (3) Taurine Post-Train (n = 20). The aim of the present study was to determine the effects of taurine given 15 min before training when compared to being given after training but 15 min before testing at 24 h on learning and memory consolidation of the AAT. The results showed in Control rats that females had shorter latencies to cross in the shuttle box, increased rates of correct learning by the % Avoids/Escapes, and decreased rates of learning errors by the % Shocks. In Taurine Post-Train male rats, taurine treatment decreased their latency to cross in the shuttle box and their rate of learning errors by the % Shocks at 24 h and 48 h Testing, but it had no effect on their rate of correct learning by the % Avoids/Escapes when compared to Control and Taurine Pre-Train male rats. In contrast, Taurine Post-Train female rats increased their latency to cross in the shuttle box during Training, 24 h and 48 h Testing, when compared to the Control and Taurine Pre-Train female rats. Further, Taurine Post-Train female rats decreased their rate of learning % Avoids/Escapes and increased the rate of learning errors % Shocks when compared to Control female rats during Training and 24 h Testing but decreased their rate of learning % Avoids/Escapes and increased the rate of learning errors % Shocks when compared to Taurine Pre-Train female rats across all test conditions. These findings suggest that neurotypical female rats may be more sensitive to the aversive stimuli (i.e., foot shocks) used in the AAT as a motivating factor for learning that may cause paradoxical behavioral learning and memory patterns. This phenomenon raises an important concern for researchers to consider when studying learning and behavioral tests in rodents that use aversive and non-aversive stimuli or a combination of both such as in the AAT. Taurine, albeit neuroprotective, may not have as much benefit in a neurotypical animal model and may increase the susceptibility for anxiogenic behaviors and interfere with cognitive learning and memory behaviors. Therefore, the mechanistic way(s) in which taurine can treat, recovery, ameliorate, and forestall other neuropathological diseases in animal models may have different psychopharmacodynamics and psychopharmacokinetics in a neurotypical animal model and should be studied with caution. This does not preclude the continued investigation of taurine psychopharmacotherapies for neuropathological diseases but encourages the careful investigation of taurine supplementation and treatment in neurotypical animals as paradoxical behavioral and cognitive outcomes have been observed herein.

Age- and sex-dependent differences in the morphology and composition of paraspinal muscles between subjects with and without lumbar degenerative diseases.

BACKGROUND: The quality of the paraspinal muscles has been recommended as a surrogate marker for the evaluation of the severity of the lumbar degenerative diseases (LDD). The purpose of this study is to determine the age- and sex-dependent differences in the morphology and composition of the paraspinal muscles between LDD and asymptomatic subjects. METHODS: We analyzed data from 370 patients and 327 asymptomatic volunteers aged between 18-85 years. The measurement of the cross-sectional area (CSA) of the erector spinae, multifidus, and psoas at the L4/5-disc level was performed by the magnetic resonance imaging (MRI). The fatty infiltration ratio (FI %) of the multifidus and erector spinae was calculated. RESULTS: FI % of the lumbar paraspinal muscles were significantly and positively correlated with the severity of LDD instead of the CSA. Males had greater CSA than females, and females showed higher FI % than males in the paraspinal muscles. With the increase of age, the CSA of the lumbar paraspinal muscles gradually decreased, and the psoas showed the most significant decreasing trend. However, the FI % gradually increased in both LDD and asymptomatic groups with aging. CONCLUSION: Age- and sex-dependent differences were found in the morphology and composition of the paraspinal muscles between subjects with and without LDD. Further long-term follow up investigations and basic studies will continue to confirm the natural history of the paraspinal muscles with aging and their association with LDD.

Connexin 43 and Connexin 26 Involvement in the Ponatinib-Induced Cardiomyopathy: Sex-Related Differences in a Murine Model.

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.

Maternal high-fat diet induces long-term obesity with sex-dependent metabolic programming of adipocyte differentiation, hypertrophy and dysfunction in the offspring.

Maternal obesity determines obesity and metabolic diseases in the offspring. The white adipose tissue (WAT) orchestrates metabolic pathways, and its dysfunction contributes to metabolic disorders in a sex-dependent manner. Here, we tested if sex differences influence the molecular mechanisms of metabolic programming of WAT in offspring of obese dams. To this end, maternal obesity was induced with high-fat diet (HFD) and the offspring were studied at an early phase [postnatal day 21 (P21)], a late phase (P70) and finally P120. In the early phase we found a sex-independent increase in WAT in offspring of obese dams using magnetic resonance imaging (MRI), which was more pronounced in females than males. While the adipocyte size increased in both sexes, the distribution of WAT differed in males and females. As mechanistic hints, we identified an inflammatory response in females and a senescence-associated reduction in the preadipocyte factor DLK in males. In the late phase, the obese body composition persisted in both sexes, with a partial reversal in females. Moreover, female offspring recovered completely from both the adipocyte hypertrophy and the inflammatory response. These findings were linked to a dysregulation of lipolytic, adipogenic and stemness-related markers as well as AMPKα and Akt signaling. Finally, the sex-dependent metabolic programming persisted with sex-specific differences in adipocyte size until P120. In conclusion, we do not only provide new insights into the molecular mechanisms of sex-dependent metabolic programming of WAT dysfunction, but also highlight the sex-dependent development of low- and high-grade pathogenic obesity.

Singularities of nevirapine metabolism: from sex-dependent differences to idiosyncratic toxicity.

Nevirapine (NVP) is a first-generation non-nucleoside reverse transcriptase inhibitor widely used for the treatment and prophylaxis of human immunodeficiency virus infection. The drug is taken throughout the patient's life and, due to the availability of an extended-release formulation, it is administered once daily. This antiretroviral is one of the scarce examples of drugs with prescription criteria based on sex, in order to prevent adverse reactions. The therapy with NVP has been associated with potentially life-threatening liver and idiosyncratic skin toxicity. Multiple evidence has emerged regarding the formation of electrophilic NVP metabolites as crucial for adverse idiosyncratic reactions. The formation of reactive metabolites that yield covalent adducts with proteins has been demonstrated in patients under NVP-based treatment. Interestingly, several pharmacogenetic- and sex-related factors associated with NVP toxicity can be mechanistically explained by an imbalance toward increased formation of NVP-derived reactive metabolites and/or impaired detoxification capability. Moreover, the haptenation of self-proteins by these reactive species provides a plausible link between NVP bioactivation and immunotoxicity, further supporting the relevance of this toxicokinetics hypothesis. In the current paper, we review the existing knowledge and recent developments on NVP metabolism and their relation to NVP toxicity.

Sex-Dependent Effects on Influenza-Specific Antibody Quantity and Neutralizing Activity following Vaccination of Newborn Non-Human Primates Is Determined by Adjuvants.

A number of studies have demonstrated the role of sex in regulating immune responses to vaccination. However, these findings have been limited to adults for both human and animal models. As a result, our understanding of the impact of sex on vaccine responses in the newborn is highly limited. Here, we probe this important question using a newborn non-human primate model. We leveraged our prior analysis of two cohorts of newborns, with one being mother-reared and one nursery-reared. This provided adequate numbers of males and females to interrogate the impact of sex on the response to inactivated influenza vaccines alone or adjuvanted with R848, flagellin, or both. We found that, in contrast to what has been reported in adults, the non-adjuvanted inactivated influenza virus vaccine induced similar levels of virus-specific IgG in male and female newborns. However, the inclusion of R848, either alone or in combination with flagellin, resulted in higher antibody titers in females compared to males. Sex-specific increases in the neutralizing antibody were only observed when both R848 and flagellin were present. These data, generated in the highly translational NHP newborn model, provide novel insights into the role of sex in the immune response of newborns.

Sex-dependent differences in animal cognition.

The differences in cognitive processes driven by biological sex are the issues that have gotten growing attention recently. Considering the increasing population suffering from various cognitive impairments and the development of therapeutic strategies, it is essential that we recognize the mechanisms responsible for discrepancies observed in male and female learning and memory functions. In this review, we discuss recent reports from preclinical studies on rodents regarding selected cognitive domains to explore the state of knowledge on sex-dependent differences and point to challenges encountered during such research. We focus on spatial, recognition, and emotional memory, as well as on executive functions, such as attention, cognitive flexibility, and working memory. This review will help to acknowledge sex-related differences in cognition and indicate some fields that lack sufficient data.

Urinary ACE Phenotyping as a Research and Diagnostic Tool: Identification of Sex-Dependent ACE Immunoreactivity.

BACKGROUND: Angiotensin-converting enzyme (ACE) is highly expressed in renal proximal tubules, but ACE activity/levels in the urine are at least 100-fold lower than in the blood. Decreased proximal tubular ACE has been associated with renal tubular damage in both animal models and clinical studies. Because ACE is shed into urine primarily from proximal tubule epithelial cells, its urinary ACE measurement may be useful as an index of tubular damage. OBJECTIVE AND METHODOLOGY: We applied our novel approach-ACE phenotyping-to characterize urinary ACE in volunteer subjects. ACE phenotyping includes (1) determination of ACE activity using two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of the two substrates (ZPHL/HHL ratio); (3) quantification of ACE immunoreactive protein levels; and (4) fine mapping of local ACE conformation with mAbs to ACE. PRINCIPAL FINDINGS: In normal volunteers, urinary ACE activity was 140-fold less than in corresponding plasma/serum samples and did not differ between males and females. However, urinary ACE immunoreactivity (normalized binding of 25 mAbs to different epitopes) was strongly sex-dependent for the several mAbs tested, an observation likely explained by differences in tissue ACE glycosylation/sialylation between males and females. Urinary ACE phenotyping also allowed the identification of ACE outliers. In addition, daily variability of urinary ACE has potential utility as a feedback marker for dieting individuals pursuing weight loss. CONCLUSIONS/SIGNIFICANCE: Urinary ACE phenotyping is a promising new approach with potential clinical significance to advance precision medicine screening techniques.

Retinal Ganglion Cells: Global Number, Density and Vulnerability to Glaucomatous Injury in Common Laboratory Mice.

How many RBPMS+ retinal ganglion cells (RGCs) does a standard C57BL/6 laboratory mouse have on average and is this number substrain- or sex-dependent? Do RGCs of (European) C57BL/6J and -N mice show a different intrinsic vulnerability upon glaucomatous injury? Global RGC numbers and densities of common laboratory mice were previously determined via axon counts, retrograde tracing or BRN3A immunohistochemistry. Here, we report the global RGC number and density by exploiting the freely available tool RGCode to automatically count RGC numbers and densities on entire retinal wholemounts immunostained for the pan-RGC marker RBPMS. The intrinsic vulnerability of RGCs from different substrains to glaucomatous injury was evaluated upon introduction of the microbead occlusion model, followed by RBPMS counts, retrograde tracing and electroretinography five weeks post-injury. We demonstrate that the global RGC number and density varies between substrains, yet is not sex-dependent. C57BL/6J mice have on average 46K ± 2K RBPMS+ RGCs per retina, representing a global RGC density of 3268 ± 177 RGCs/mm2. C57BL/6N mice, on the other hand, have on average less RBPMS+ RGCs (41K ± 3K RGCs) and a lower density (3018 ± 189 RGCs/mm2). The vulnerability of the RGC population of the two C57BL/6 substrains to glaucomatous injury did, however, not differ in any of the interrogated parameters.

Influence of Age and Sex on Disease Course and Treatment in Rheumatoid Arthritis.

OBJECTIVE: More than 50% of patients with rheumatoid arthritis (RA) are >65 years at diagnosis. Age of onset and sex may influence the disease course, outcome and treatment. This study follows a large cohort of patients with early RA to assess contributions of age and sex to disease outcomes. METHODS: Patients from the BARFOT cohort, n=2837 (68% women), were followed for eight years at predefined time points to assess inflammation, function, joint destruction and treatment with disease modifying anti-rheumatic drugs (DMARDs) and glucocorticoids (GC). The patients were divided by sex and age at inclusion (<40, 40-54, 55-69 and ≥70 years). RESULTS: For both sexes, disease activity, function and pain improved over time, significantly more in men than in women in all age groups. In men, those <40 years displayed significantly lower DAS28 compared with all other groups. This group was also the least represented group in the study. The Sharp van der Heijde Score (SHS) increased over time in both sexes and all age groups. Women ≥70 years showed less improvement in disability and the highest progression of SHS mainly due to increased joint space narrowing. Patients <40 years were more likely to receive biological DMARDs, while those ≥70 years more often received only GC treatment. CONCLUSION: There were significant age- and sex-dependent differences in the medical treatment and in outcome of RA 8 years after diagnosis. The differences were most pronounced in men<40 and women ≥70 years, but whether they are due to disease phenotype or treatment is unclear.

Early life adversity affecting the attachment bond alters ventral tegmental area transcriptomic patterning and behavior almost exclusively in female mice.

Early life experiences that affect the attachment bond formation can alter developmental trajectories and result in pathological outcomes in a sex-related manner. However, the molecular basis of sex differences is quite unknown. The dopaminergic system originating from the ventral tegmental area has been proposed to be a key mediator of this process. Here we exploited a murine model of early adversity (Repeated Cross Fostering, RCF) to test how interfering with the attachment bond formation affects the VTA-related functions in a sex-specific manner. Through a comprehensive behavioral screening, within the NiH RDoC framework, and by next-generation RNA-Seq experiments, we analyzed the long-lasting effect of RCF on behavioral and transcriptional profiles related to the VTA, across two different inbred strains of mouse in both sexes. We found that RCF impacted to an extremely greater extent VTA-related behaviors in females than in males and this result mirrored the transcriptional alterations in the VTA that were almost exclusively observed in females. The sexual dimorphism was conserved across two different inbred strains in spite of their divergent long lasting consequences of RCF exposure. Our data suggest that to be female primes a sub-set of genes to respond to early environmental perturbations. This is, to the best of our knowledge, the first evidence of an almost exclusive effect of early life experiences on females, thus mirroring the extremely stronger impact of precocious aversive events reported in clinical studies in women.

Aging and aging-associated diseases: a microRNA-based endocrine regulation hypothesis.

Although there are numerous hypotheses explaining the nature of aging and associated processes, two concepts are dominant: (i) aging is a result of cell-autonomous processes, such as the accumulation of DNA mutations, aberrant methylations, protein defects, and shortening of telomeres, leading to either inhibition of cellular proliferation and death of non-dividing terminally differentiated cells or tumor development; (ii) aging is a result of a central program that is switched on at a specific stage of organismic development. The microRNA-based endocrine regulation hypothesis combines the two above concepts by proposing central regulation of cell death occurrences via hypothalamus-pituitary gland (PG)-secreted miRNA hormones, the expression and/or secretion of which are regulated by sex hormones. This hypothesis explains such well-known phenomena as inverse comorbidity of either cancer or Alzheimer's (AD) and other neurodegenerative diseases; higher AD morbidity and lower frequency of many common types of cancer in women vs. men; higher risk of early AD and lower risk of cancer in subjects with Down syndrome; longer life expectancy in women vs. men and much lower sex-dependent differences, if any, in other mammals; increased lifespans due to hypophysectomy or PG hypofunction; and parabiotic effects of blood or plasma transfusions between young and old animals.

Effect of voluntary wheel running on neuroactive steroid levels in murine experimental autoimmune encephalomyelitis.

Increasing evidence from both clinical and animal research has implicated changes in neuroactive steroids (rapid acting steroids that act as allosteric modulators at NMDA and/or GABA-A receptors) in multiple sclerosis. These changes have been linked to clinical differences in disease severity, prevention of disease development, as well as the disease state (relapsing vs progressive) in patients with multiple sclerosis. Previous research has also linked changes in neuroactive steroid levels to the beneficial effects of exercise in certain disorders such as traumatic brain injury and post-traumatic stress disorder. The present study therefore examined whether voluntary wheel running could modulate any of the reported changes in neuroactive steroids associated with the EAE model of multiple sclerosis. Female mice with EAE who ran were found to have significantly increased levels of brain pregnenolone compared to male EAE mice who ran. In contrast, male mice with EAE were found to have significantly higher levels of brain allopregnanolone compared to female mice with EAE regardless of exercise. Overall, these results indicate that exercise has moderate beneficial effects on brain neuroactive steroid levels in EAE. These changes may be related to other beneficial responses to exercise, such as improvements in disease severity, in EAE and/or multiple sclerosis.