Angiotensin-(1-12): Does It Exist? A Critical Evaluation in Humans, Rats, and Mice.

BACKGROUND: Angiotensin-(1-12), measured by a self-developed, polyclonal antibody-based radioimmunoassay, has been suggested to act as an alternative precursor of angiotensin II. A more reliable detection method would be liquid chromatography-tandem mass spectrometry. METHODS: We set up the quantification of human and murine angiotensin-(1-12) by liquid chromatography-tandem mass spectrometry and then used this method to measure angiotensin-(1-12) in human and mouse blood samples, as well as in mouse brain and kidney. We also verified ex vivo angiotensin-(1-12) generation and metabolism in human blood samples incubated at 37 °C. RESULTS: Stabilization of blood in guanidine hydrochloride was chosen for sample collection since this allowed full recovery of spiked angiotensin-(1-12). Angiotensin-(1-12) was undetectable in human blood samples when incubating nonstabilized plasma at 37 °C, while angiotensin-(1-12) added to nonstabilized human plasma disappeared within 10 minutes. Stabilized human blood samples contained angiotensin II, while angiotensin-(1-12) was undetectable. Blood, hearts, and kidneys, but not brains, of wild-type mice and rats contained detectable levels of angiotensin II, while angiotensin-(1-12) was undetectable. In renin knockout mice, all angiotensins, including angiotensin-(1-12), were undetectable at all sites, despite a 50% rise in angiotensinogen. Angiotensin-(1-12) metabolism in human blood plasma was not affected by renin inhibition. Yet, blockade of angiotensin-converting enzyme and aminopeptidase A, but not of chymase, neutral endopeptidase, or prolyl oligopeptidase, prolonged the half-life of angiotensin-(1-12), and angiotensin-converting enzyme inhibition prevented the formation of angiotensin II. CONCLUSIONS: We were unable to detect intact angiotensin-(1-12) in humans or mice, either in blood or tissue, suggesting that this metabolite is an unlikely source of endogenous angiotensins.

Elevated sortilin expression discriminates functional from non-functional neuroendocrine tumors and enables therapeutic targeting.

A subset of neuroendocrine tumors (NETs) can cause an excessive secretion of hormones, neuropeptides, and biogenic amines into the bloodstream. These so-called functional NETs evoke a hormone-related disease and lead to several different syndromes, depending on the factors released. One of the most common functional syndromes, carcinoid syndrome, is characterized mainly by over-secretion of serotonin. However, what distinguishes functional from non-functional tumors on a molecular level remains unknown. Here, we demonstrate that the expression of sortilin, a widely expressed transmembrane receptor involved in intracellular protein sorting, is significantly increased in functional compared to non-functional NETs and thus can be used as a biomarker for functional NETs. Furthermore, using a cell line model of functional NETs, as well as organoids, we demonstrate that inhibition of sortilin reduces cellular serotonin concentrations and may therefore serve as a novel therapeutic target to treat patients with carcinoid syndrome.

Poor Decision Making and Sociability Impairment Following Central Serotonin Reduction in Inducible TPH2-Knockdown Rats.

Serotonin is an essential neuromodulator for mental health and animals' socio-cognitive abilities. However, we previously found that a constitutive depletion of central serotonin did not impair rat cognitive abilities in stand-alone tests. Here, we investigated how a mild and acute decrease in brain serotonin would affect rats' cognitive abilities. Using a novel rat model of inducible serotonin depletion via the genetic knockdown of tryptophan hydroxylase 2 (TPH2), we achieved a 20% decrease in serotonin levels in the hypothalamus after three weeks of non-invasive oral doxycycline administration. Decision making, cognitive flexibility, and social recognition memory were tested in low-serotonin (Tph2-kd) and control rats. Our results showed that the Tph2-kd rats were more prone to choose disadvantageously in the long term (poor decision making) in the Rat Gambling Task and that only the low-serotonin poor decision makers were more sensitive to probabilistic discounting and had poorer social recognition memory than other low-serotonin and control individuals. Flexibility was unaffected by the acute brain serotonin reduction. Poor social recognition memory was the most central characteristic of the behavioral network of low-serotonin poor decision makers, suggesting a key role of social recognition in the expression of their profile. The acute decrease in brain serotonin appeared to specifically amplify the cognitive impairments of the subgroup of individuals also identified as poor decision makers in the population. This study highlights the great opportunity the Tph2-kd rat model offers to study inter-individual susceptibilities to develop cognitive impairment following mild variations of brain serotonin in otherwise healthy individuals. These transgenic and differential approaches together could be critical for the identification of translational markers and vulnerabilities in the development of mental disorders.

Role of kinin receptors in skin pigmentation.

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Karyopherin α2 is a maternal effect gene required for early embryonic development and female fertility in mice.

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene.

Biotransformation and toxicokinetics of 2-phenoxyethanol after oral exposure in humans: a volunteer study.

2-Phenoxyethanol (PhE) is an aromatic glycol ether and is used in a variety of functions and applications, e.g., as preservative in pharmaceuticals, cosmetic and personal care products, as biocide in disinfectants (e.g. human hygiene), or as a solvent in formulations (e.g. coatings, functional fluids). Despite its widespread use, little is yet known on its biotransformation and toxicokinetics in humans. Therefore, a pilot study was conducted with oral administration of PhE (5 mg/kg body weight) to five volunteers. Blood and urine samples were collected and analyzed for PhE and three of its presumed metabolites up to 48 h post-exposure. Additionally, one volunteer was dermally exposed to PhE and monitored until 72 h post-exposure. PhE was rapidly resorbed following both oral and dermal application with tmax-levels in blood of about 1 h and 3 h, respectively. Metabolism of PhE was observed to be rather extensive with phenoxyacetic acid (PhAA) and 4-hydroxyphenoxyacetic acid (4-OH-PhAA) as the main metabolites found in blood and urine following oral and dermal exposure. PhE was excreted rapidly and efficiently via urine mostly in metabolized form: following oral exposure, on average 77% and 12% of the applied dose was excreted within 48 h as PhAA and 4-OH-PhAA, respectively. A similar metabolism pattern was observed following the single dermal exposure experiment. The obtained data on biotransformation and toxicokinetics of PhE in humans provide valuable information on this important chemical and will be highly useful for pharmacokinetic modelling and evaluation of human PhE exposure.

A specific and sensitive GC-MS/MS method for the quantitative determination of 2-phenoxyethanol and selected metabolites in human blood and urine.

2-Phenoxyethanol (PhE) is widely used as a preservative in consumer products such as cosmetics as well as at the workplace as a component of metal-working fluids and hydraulic fluids. Therefore, both industry workers and consumers may potentially be exposed to PhE. An analytical method for the quantification of PhE and three selected metabolites, namely phenoxyacetic acid (PhAA), 4-hydroxyphenoxyacetic acid (4-OH-PhAA), and 4-hydroxyphenoxyethanol (4-OH-PhE) in human urine and blood was developed and validated. The sample preparation includes enzymatic hydrolysis of urine samples or protein precipitation of blood samples, followed by liquid-liquid extraction and silylation of the target analytes. Analyses of the extracts were carried out by gas chromatography with tandem mass spectrometry (GC-MS/MS). 3,4-Hydroxyphenoxyethanol, a probably minor PhE metabolite could not reliable be analyzed due to its instability. The limits of quantification (LOQ) of the analytes ranged between 0.5 and 6.1 µg/L and 2.0 and 3.9 µg/L in urine and blood, respectively. The method was successfully applied to spot urine samples of 50 individuals without occupational exposure to PhE and additionally to blood samples from seven volunteers. In urine, PhAA and 4-OH-PhAA could be quantified in all analyzed samples, whereas 4-OH-PhE and unchanged PhE were found in 36 % and 32 % of the samples, respectively. In blood, PhAA was also found in every sample in levels above the LOQ, whereas PhE itself was detected in three of seven samples only. Neither 4-OH-PhAA nor 4-OH-PhE were found in any of the analyzed blood samples. The developed method promises to be a valuable tool for PhE monitoring of urine and blood samples and may also enable an advanced investigation of PhE biotransformation pathways in humans.

Tumor associated microglia/macrophages utilize GPNMB to promote tumor growth and alter immune cell infiltration in glioma.

Tumor-associated microglia and blood-derived macrophages (TAMs) play a central role in modulating the immune suppressive microenvironment in glioma. Here, we show that GPNMB is predominantly expressed by TAMs in human glioblastoma multiforme and the murine RCAS-PDGFb high grade glioma model. Loss of GPNMB in the in vivo tumor microenvironment results in significantly smaller tumor volumes and generates a pro-inflammatory innate and adaptive immune cell microenvironment. The impact of host-derived GPNMB on tumor growth was confirmed in two distinct murine glioma cell lines in organotypic brain slices from GPNMB-KO and control mice. Using published data bases of human glioma, the elevated levels in TAMs could be confirmed and the GPNMB expression correlated with a poorer survival.

Anxiolytic effect of alamandine in male transgenic rats with low brain angiotensinogen is dependent on activation of MrgD receptors.

Alamandine is a peptide hormone belonging to the renin-angiotensin system (RAS). It acts through the Mas-related G-protein coupled receptor type D, MrgD, which is expressed in different tissues, including the brain. In the present study, we hypothesize that a lack of alamandine, through MrgD, could cause the anxiety-like behavior in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)680]. Adult male transgenic rats exhibited a significant increase in the latency to feeding time in the novelty suppressed feeding test and a decrease in the percentage of time and entries in the open arms in the elevated plus maze. These effects were reversed by intracerebroventricular infusion of alamandine. Pretreatment with D-Pro7-Ang-(1-7), a Mas and MrgD receptor antagonist, prevented the anxiolytic effects induced by this peptide. However, its effects were not altered by the selective Mas receptor antagonist, A779. In conclusion, our data indicates that alamandine, through MrgD, attenuates anxiety-like behavior in male TGR(ASrAOGEN)680, which reinforces the importance of the counter-regulatory RAS axis as promising target for the treatment of neuropsychiatric disorders.

Evaluation of a Condition Monitoring Algorithm for Early Bearing Fault Detection.

Roller bearings are critical components in various mechanical systems, and the timely detection of potential failures is essential for preventing costly downtimes and avoiding substantial machinery breakdown. This research focuses on finding and verifying a robust method that can detect failures early, without creating false positive failure states. Therefore, this paper introduces a novel algorithm for the early detection of roller bearing failures, particularly tailored to high-precision bearings and automotive test bed systems. The featured method (AFI-Advanced Failure Indicator) utilizes the Fast Fourier Transform (FFT) of wideband accelerometers to calculate the spectral content of vibration signals emitted by roller bearings. By calculating the frequency bands and tracking the movement of these bands within the spectra, the method provides an indicator of the machinery's health, mainly focusing on the early stages of bearing failure. The calculated channel can be used as a trend indicator, enabling the method to identify subtle deviations associated with impending failures. The AFI algorithm incorporates a non-static limit through moving average calculations and volatility analysis methods to determine critical changes in the signal. This thresholding mechanism ensures the algorithm's responsiveness to variations in operating conditions and environmental factors, contributing to its robustness in diverse industrial settings. Further refinement was achieved through an outlier detection filter, which reduces false positives and enhances the algorithm's accuracy in identifying genuine deviations from the normal operational state. To benchmark the developed algorithm, it was compared with three industry-standard algorithms: VRMS calculations per ISO 10813-3, Mean Absolute Value of Extremums (MAVE), and Envelope Frequency Band (EFB). This comparative analysis aimed to evaluate the efficacy of the novel algorithm against the established methods in the field, providing valuable insights into its potential advantages and limitations. In summary, this paper presents an innovative algorithm for the early detection of roller bearing failures, leveraging FFT-based spectral analysis, trend monitoring, adaptive thresholding, and outlier detection. Its ability to confirm the first failure state underscores the algorithm's effectiveness.

Alternative Renin-Angiotensin System.

The renin-angiotensin system is the most important peptide hormone system in the regulation of cardiovascular homeostasis. Its classical arm consists of the enzymes, renin, and angiotensin-converting enzyme, generating angiotensin II from angiotensinogen, which activates its AT1 receptor, thereby increasing blood pressure, retaining salt and water, and inducing cardiovascular hypertrophy and fibrosis. However, angiotensin II can also activate a second receptor, the AT2 receptor. Moreover, the removal of the C-terminal phenylalanine from angiotensin II by ACE2 (angiotensin-converting enzyme 2) yields angiotensin-(1-7), and this peptide interacts with its receptor Mas. When the aminoterminal Asp of angiotensin-(1-7) is decarboxylated, alamandine is generated, which activates the Mas-related G-protein-coupled receptor D, MrgD (Mas-related G-protein-coupled receptor type D). Since Mas, MrgD, and the AT2 receptor have opposing effects to the classical AT1 receptor, they and the enzymes and peptides activating them are called the alternative or protective arm of the renin-angiotensin system. This review will cover the historical aspects and the current standing of this recent addition to the biology of the renin-angiotensin system.

Effects of Bradykinin B2 Receptor Ablation from Tyrosine Hydroxylase Cells on Behavioral and Motor Aspects in Male and Female Mice.

The kallikrein-kinin system is a versatile regulatory network implicated in various biological processes encompassing inflammation, nociception, blood pressure control, and central nervous system functions. Its physiological impact is mediated through G-protein-coupled transmembrane receptors, specifically the B1 and B2 receptors. Dopamine, a key catecholamine neurotransmitter widely distributed in the CNS, plays a crucial role in diverse physiological functions including motricity, reward, anxiety, fear, feeding, sleep, and arousal. Notably, the potential physical interaction between bradykinin and dopaminergic receptors has been previously documented. In this study, we aimed to explore whether B2R modulation in catecholaminergic neurons influences the dopaminergic pathway, impacting behavioral, metabolic, and motor aspects in both male and female mice. B2R ablation in tyrosine hydroxylase cells reduced the body weight and lean mass without affecting body adiposity, substrate oxidation, locomotor activity, glucose tolerance, or insulin sensitivity in mice. Moreover, a B2R deficiency in TH cells did not alter anxiety levels, exercise performance, or motor coordination in female and male mice. The concentrations of monoamines and their metabolites in the substantia nigra and cortex region were not affected in knockout mice. In essence, B2R deletion in TH cells selectively influenced the body weight and composition, leaving the behavioral and motor aspects largely unaffected.

Serotonin Transporter-dependent Histone Serotonylation in Placenta Contributes to the Neurodevelopmental Transcriptome.

Brain development requires appropriate regulation of serotonin (5-HT) signaling from distinct tissue sources across embryogenesis. At the maternal-fetal interface, the placenta is thought to be an important contributor of offspring brain 5-HT and is critical to overall fetal health. Yet, how placental 5-HT is acquired, and the mechanisms through which 5-HT influences placental functions, are not well understood. Recently, our group identified a novel epigenetic role for 5-HT, in which 5-HT can be added to histone proteins to regulate transcription, a process called H3 serotonylation. Here, we show that H3 serotonylation undergoes dynamic regulation during placental development, corresponding to gene expression changes that are known to influence key metabolic processes. Using transgenic mice, we demonstrate that placental H3 serotonylation is dependent on 5-HT uptake by the serotonin transporter (SERT/SLC6A4). SERT deletion robustly reduces enrichment of H3 serotonylation across the placental genome, and disrupts neurodevelopmental gene networks in early embryonic brain tissues. Thus, these findings suggest a novel role for H3 serotonylation in coordinating placental transcription at the intersection of maternal physiology and offspring brain development.

IL-1ß promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells.

ABSTRACT: JAK 2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1ß (IL-1ß)-mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1ß-/- or IL-1R1-/- mice. To study the role of IL-1ß and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1ß in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1ß levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1ß levels, and displayed low GFP-chimerism resembling CHIP. Anti-IL-1ß antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1ß as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

Kinin B1 receptor modulates glucose homeostasis and physical exercise capacity by altering adrenal catecholamine synthesis and secretion.

Our group has shown in several papers that kinin B1 receptor (B1R) is involved in metabolic adaptations, mediating glucose homeostasis and interfering in leptin and insulin signaling. Since catecholamines are involved with metabolism management, we sought to evaluate B1R role in catecholamine synthesis/secretion. Using B1R global knockout mice, we observed increased basal epinephrine content, accompanied by decreased hepatic glycogen content and increased glucosuria. When these mice were challenged with maximal intensity exercise, they showed decreased epinephrine and norepinephrine response, accompanied by disturbed glycemic responses to effort and poor performance. This phenotype was related to alterations in adrenal catecholamine synthesis: increased basal epinephrine concentration and reduced norepinephrine content in response to exercise, as well decreased gene expression and protein content of tyrosine hydroxylase and decreased gene expression of dopamine beta hydroxylase and kinin B2 receptor. We conclude that the global absence of B1R impairs catecholamine synthesis, interfering with glucose metabolism at rest and during maximal exercise.

Kinin B1 receptor controls maternal adiponectin levels and influences offspring weight gain.

Given the importance of the kinin B1 receptor in insulin and leptin hormonal regulation, which in turn is crucial in maternal adaptations to ensure nutrient supply to the fetus, we investigated the role of this receptor in maternal metabolism and fetoplacental development. Wild-type and kinin B1 receptor-deficient (B1KO) female mice were mated with male mice of the opposite genotype. Consequently, the entire litter was heterozygous for kinin B1 receptor, ensuring that there would be no influence of offspring genotype on the maternal phenotype. Maternal kinin B1 receptor blockade reduces adiponectin secretion by adipose tissue ex vivo, consistent with lower adiponectin levels in pregnant B1KO mice. Furthermore, fasting insulinemia also increased, which was associated with placental insulin resistance, reduced placental glycogen accumulation, and heavier offspring. Therefore, we propose the combination of chronic hyperinsulinemia and reduced adiponectin secretion in B1KO female mice create a maternal obesogenic environment that results in heavier pups.

GPER activation attenuates cardiac dysfunction by upregulating the SIRT1/3-AMPK-UCP2 pathway in postmenopausal diabetic rats.

Postmenopausal diabetic women are at higher risk to develop cardiovascular diseases (CVD) compared with nondiabetic women. Alterations in cardiac cellular metabolism caused by changes in sirtuins are one of the main causes of CVD in postmenopausal diabetic women. Several studies have demonstrated the beneficial actions of the G protein-coupled estrogen receptor (GPER) in postmenopausal diabetic CVD. However, the molecular mechanisms by which GPER has a cardioprotective effect are still not well understood. In this study, we used an ovariectomized (OVX) type-two diabetic (T2D) rat model induced by high-fat diet/streptozotocin to investigate the effect of G-1 (GPER-agonist) on sirtuins, and their downstream pathways involved in regulation of cardiac metabolism and function. Animals were divided into five groups: Sham-Control, T2D, OVX+T2D, OVX+T2D+Vehicle, and OVX+T2D+G-1. G-1 was administrated for six weeks. At the end, hemodynamic factors were measured, and protein levels of sirtuins, AMP-activated protein kinase (AMPK), and uncoupling protein 2 (UCP2) were determined by Western blot analysis. In addition, cardiac levels of oxidative stress biomarkers were measured. The findings showed that T2D led to left ventricular dysfunction and signs of oxidative stress in the myocardium, which were accompanied by decreased protein levels of Sirt1/2/3/6, p-AMPK, and UCP2 in the heart. Moreover, the induction of the menopausal state exacerbated these changes. In contrast, treatment with G-1 ameliorated the hemodynamic changes associated with ovariectomy by increasing Sirt1/3, p-AMPK, UCP2, and improving oxidative status. The results provide evidence of the cardioprotective effects of GPER operating through Sirt1/3, p-AMPK, and UCP2, thereby improving cardiac function. Our results suggest that increasing Sirt1/3 levels may offer new therapeutic approaches for postmenopausal diabetic CVD.

Serotonin transporter-dependent histone serotonylation in placenta contributes to the neurodevelopmental transcriptome.

Brain development requires appropriate regulation of serotonin (5-HT) signaling from distinct tissue sources across embryogenesis. At the maternal-fetal interface, the placenta is thought to be an important contributor of offspring brain 5-HT and is critical to overall fetal health. Yet, how placental 5-HT is acquired, and the mechanisms through which 5-HT influences placental functions, are not well understood. Recently, our group identified a novel epigenetic role for 5-HT, in which 5-HT can be added to histone proteins to regulate transcription, a process called H3 serotonylation. Here, we show that H3 serotonylation undergoes dynamic regulation during placental development, corresponding to gene expression changes that are known to influence key metabolic processes. Using transgenic mice, we demonstrate that placental H3 serotonylation largely depends on 5-HT uptake by the serotonin transporter (SERT/SLC6A4). SERT deletion robustly reduces enrichment of H3 serotonylation across the placental genome, and disrupts neurodevelopmental gene networks in early embryonic brain tissues. Thus, these findings suggest a novel role for H3 serotonylation in coordinating placental transcription at the intersection of maternal physiology and offspring brain development.