Chronic Behavioral Stress Research Articles

Hypertension and renal disease programming: focus on the early postnatal period.

The developmental origin of hypertension and renal disease is a concept highly supported by strong evidence coming from both human and animal studies. During development there are periods in which the organs are more vulnerable to stressors. Such periods of susceptibility are also called 'sensitive windows of exposure'. It was shown that as earlier an adverse event occurs; the greater are the consequences for health impairment. However, evidence show that the postnatal period is also quite important for hypertension and renal disease programming, especially in rodents because they complete nephrogenesis postnatally, and it is also important during preterm human birth. Considering that the developing kidney is vulnerable to early-life stressors, renal programming is a key element in the developmental programming of hypertension and renal disease. The purpose of this review is to highlight the great number of studies, most of them performed in animal models, showing the broad range of stressors involved in hypertension and renal disease programming, with a particular focus on the stressors that occur during the early postnatal period. These stressors mainly include undernutrition or specific nutritional deficits, chronic behavioral stress, exposure to environmental chemicals, and pharmacological treatments that affect some important factors involved in renal physiology. We also discuss the common molecular mechanisms that are activated by the mentioned stressors and that promote the appearance of these adult diseases, with a brief description on some reprogramming strategies, which is a relatively new and promising field to treat or to prevent these diseases.

Pharmacologic inhibition of beta-adrenergic receptors decreases PD-L1 mediated immunosuppression and improves anti-tumor immune signature in ovarian cancer (126)

<b>Objectives:</b> The immune system is affected by stress hormones, but also plays an important role in activating and suppressing mechanisms controlling epithelial ovarian cancer (EOC). Although EOC is a "cold tumor" the trafficking of tumor infiltrating lymphocytes in the tumor mass and the expression of programmed cell death ligand (PD-L1) are prognostic indicators in EOC. To date PD-1/PD-L1 inhibitors have had limited benefit as single agents or in combination with chemotherapy. This study aimed to explore the impact of adrenergic stress and its blockade with propranolol in both <i>in vitro</i> and <i>in vivo</i> EOC models. <b>Methods:</b> Two high grade serous cell lines SKOV3ip1 and OVCAR8 were used in co-culture experiments and exosome experiments. Western blot was used to detect changes in PD-L1 protein expression in exosomes treated with noradrenaline, propranolol, and interferon-gamma (IFN-g). C57bl/6 mice were injected with ID8 cells and exposed to chronic behavioral stress to activate the beta-adrenergic signalling pathway. Mice were treated with the following combinations: control (saline), propranolol (2mg/Kg/d via Alzet pump for 6 weeks), PD-1/PD-L1 inhibitor (200μg/mouse IP, three times a week for two weeks) or a combination of propranolol and PD-1/PD-L1 inhibitor. CD3, CD4, CD8 and CXCL10 mRNA expression were evaluated in the tumors with qPCR. GraphPad Prism v.7 was used as well as one-way ANOVA with Bonferroni multiple comparison test correction. <b>Results:</b> EOC cell lines were treated with noradrenaline (NA), propranolol, IFN-g or in combination (NA+ IFN-g). NA+IFN-g caused an increase in PD-L1 mRNA and protein expression (p<0.05). IFN-g causes a statistically significant increase in the production of PD-L1 carrying exosomes (p<0.05) while propranolol does not. Combination of propranolol and PD-1/PD-L1 inhibitor significantly reduced tumour weight compared to the cancer control group (p<0.05). Chronic behavioural stress significantly increased the number of organs with metastatic nodules (p<0.05) while propranolol monotherapy and the combined therapy showed a significant decrease in the number of organs affected by metastatic nodules (p<0.001). VEGF expression was significantly reduced in the combined therapy group compared to the propranolol and PD-1/PD-L1 monotherapy (p<0.001). CD8 CD3, CD4 mRNA expression increased significantly in the combined therapy group compared to PD-1/PD-L1 inhibitor monotherapy and the control group (p<0.05). Propranolol also decreased IDO1 and CXCL10 mRNA as well. <b>Conclusions:</b> Propranolol decreases upstream production of IFN-g and the IFN-g-mediated PD-L1 expression on cancer cells. Therefore the combination of propranolol with PD-L1 inhibitors is a promising therapy in this preclinical EOC model.

Early transcriptional response of human ovarian and fallopian tube surface epithelial cells to norepinephrine

Evidence from human and animal studies suggests that chronic behavioral stress and resulting activation of the sympathetic nervous system may influence initiation and progression of tumors. However, the underlying mechanisms for these observations are poorly understood. The purpose of this study is to explore the effects of adrenergic signaling on cell line models derived from normal cells presumed to originate epithelial ovarian cancers. Here we explored the effects of the stress-related hormone, norepinephrine, on the transcriptional program of normal immortalized ovarian (iOSE) and fallopian tube (iFTSEC) surface epithelial cells. Analysis of RNA-Seq data of treated and untreated cells revealed a significant overlap between the responses in iOSE and iFTSEC cells. Most genes modulated by norepinephrine in ovarian and fallopian tube epithelial cells are already expressed in normal ovarian and fallopian tissue and cells. For several genes, expression changes were reflected at the protein level. Genes in immune-related and developmental pathways were enriched in the set of genes modulated by norepinephrine. We identified HOXA5, SPIB, REL, SRF, SP1, NFKB1, MEF2A, E2F1, and EGR1 transcription factor binding sites to be highly enriched in our dataset. These data represent the early transcriptional response to norepinephrine in cells postulated to originate epithelial ovarian cancer.

Effects of chronic restraint stress on the global DNA methylation profile of rat lung cells: Modulation by physical exercise

The potential of behavioral stress to affect epigenetic mechanisms of non-encephalic tissues is still underestimated. In the present study we evaluated the effects of chronic behavioral stress on the DNA methylation profile of rat lung cells. Furthermore, we evaluated the potential of physical exercise to modulate the changes evoked by behavioral stress in lung cells. Male Wistar rats were divided into four experimental groups: (1) animals submitted to chronic restraint stress (CRS) (ST group) during the period of the 67th-80th postnatal day (PND); (2) animals submitted to physical exercise (EX group) during the 53rd-79th PND; (3) animals submitted to swimming during the 53rd-79th PND and to CRS during the 67th-80th PND (EX-ST group); and (4) animals not submitted to stress or swimming protocols (CTL). Global DNA methylation was quantified using an ELISA-based approach and gene expression was evaluated by real time PCR. A decreased global DNA methylation profile was observed in the ST group, however physical exercise demonstrated protection of lung cells from this stress-related hypomethylation. Increased expression of the Dnmt1 gene was evidenced in the ST group, whereas physical exercise was shown to protect lung cells from this stress-related effect in the EX-ST group. Comparative analysis of the ST and EX groups revealed opposite effects on the expression of Dnmt3a and Dnmt3b; however, a stress-related increase in expression of Dnmt3a and Dnmt3b was not seen in the EX-ST group. Our data showed that behavioral stress induced significant changes in the DNA methylation profile of rat lung cells and that this could be modulated by physical exercise.

Beta-adrenergic signaling promotes tumor angiogenesis and prostate cancer progression through HDAC2-mediated suppression of thrombospondin-1.

Chronic behavioral stress and beta-adrenergic signaling have been shown to promote cancer progression, whose underlying mechanisms are largely unclear, especially the involvement of epigenetic regulation. Histone deacetylase-2 (HDAC2), an epigenetic regulator, is critical for stress-induced cardiac hypertrophy. It is unknown whether it is necessary for beta-adrenergic signaling-promoted cancer progression. Using xenograft models, we showed that chronic behavioral stress and beta-adrenergic signaling promote angiogenesis and prostate cancer progression. HDAC2 was induced by beta-adrenergic signaling in vitro and in mouse xenografts. We next uncovered that HDAC2 is a direct target of cAMP response element-binding protein (CREB) that is activated by beta-adrenergic signaling. Notably, HDAC2 is necessary for beta-adrenergic signaling to induce angiogenesis. We further demonstrated that, upon CREB activation, HDAC2 represses thrombospondin-1 (TSP1), a potent angiogenesis inhibitor, through epigenetic regulation. Together, these data establish a novel pathway that HDAC2 and TSP1 act downstream of CREB activation in beta-adrenergic signaling to promote cancer progression.

Chronic behavioral stress exaggerates motor deficit and neuroinflammation in the MPTP mouse model of Parkinson's disease.

Environmental stressor exposure is associated with a variety of age-related diseases including neurodegeneration. Although the initial events of sporadic Parkinson's disease (PD) are not known, consistent evidence supports the hypothesis that the disease results from the combined effect of genetic and environmental risk factors. Among them, behavioral stress has been shown to cause damage and neuronal loss in different areas of the brain, however, its effect on the dopaminergic system and PD pathogenesis remains to be characterized. The C57BL/6 mice underwent chronic restraint/isolation (RI) stress and were then treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas the control mice were treated only with MPTP and the effect on the PD-like phenotype was evaluated. The mice that underwent RI before the administration of MPTP manifested an exaggerated motor deficit and impairment in the acquisition of motor skills, which were associated with a greater loss of neuronal tyrosine hydroxylase and astrocytes activation. By showing that RI influences the onset and progression of the PD-like phenotype, our study underlines the novel pathogenetic role that chronic behavioral stressor has in the disease process by triggering neuroinflammation and degeneration of the nigral dopaminergic system.

Abstract 628: Early Life Stress Exacerbates The Adrenergic Receptor Subtypes Mrna Expression In The Kidney

Maternal separation (MS) in rodents is a model of human chronic behavioral stress during early life. Previously we have shown that renal sympathetic system is sensitized in adult MS rats. Accordingly, we found that reduced glomerular filtration rate in intact MS rats was normalized after bilateral renal denervation (DnX). The aims of this study were to test the mRNA expression of adrenergic receptor (AR) subtypes in intact and DnX renal cortex and renal vessels. Also, to test the levels of mediators related to the alpha (1,4,5-trisphosphate, IP3) and beta (adenosine-3′,5′-cyclic monophosphate, cAMP) AR signaling pathways. MS was performed in WKY by separating approx half of the male pups from the dam 3 hr a day from days 2-14 of life. Renal tissue from 12 weeks old rats was harvested and snapped frozen. RT-PCR was performed using primers for ARs and IP3 receptor 1 and 2 and mRNA levels. cAMP was determined in renal cortex homogenates by ELISA. IP3 receptor mRNA expression was not different between MS and control rats in intact cortex, Dnx cortex or kidney vessels. cAMP was increased in intact renal cortex of MS rats compared to control (2.37±0.23 vs. 1.55±0.0.24 pmol/mg protein, respectively, p&lt;0.05). Relative mRNA expression (2 - ΔΔ ct ) of α1a, α1b, α1d and α2b were not different in intact cortex from control and MS rats, while Dnx did not have any significant effect. However, α2c AR was increased in intact renal cortex from MS rats compared to control rats (4.5±1.2 vs. 1.8±0.3 2 - ΔΔ ct , p&lt;0.05). DnX significantly reduced α2c mRNA expression in renal cortex from MS and control rats (0.9±0.2 and 0.5±0.1 2 - ΔΔ ct , p&lt;0.05 vs. intact cortex). Relative mRNA expression of α1a, α1b, α1d, α2b and α2c were significantly increased in renal vessels tissue from MS rats compared to control rats (p&lt;0.05). We conclude that enhanced renal mRNA expression of ARs, especially α2c, may be a key mechanisms contributing to the lower glomerular filtration capacity in MS rats. Distinctive pharmacodynamic properties of α2c ARs suggest that they may be attractive targets for therapeutic intervention in case of exposition to early life stress.

Maternal Separation (MS) enhances angiotensin II (Ang II)‐induced hypertension in Dahl rats fed a high salt diet

Previously, we showed that MS, a model of early life stress, enhances the response to hypertensive stimuli in a non‐salt sensitive rat strain. The aims of this study were to test whether MS in Dahl salt‐sensitive (DS) rats exaggerates: 1) the acute or chronic behavioral stress response, or, 2) AngII and/or high salt diet‐induced hypertension. MS was performed in male DS rats by separating about half of the pups from the dam 3 hr a day from days 2–14 after birth. Undisturbed littermates served as controls (C). Rats were implanted with telemetry transmitters (10 weeks of age, n=4–6) and allowed to recover. Mean arterial pressure (MAP) was not different between C and MS rats at baseline (118±1 vs. 120±2 mmHg). Acute air jet stress‐induced pressor response measured as area under the curve (AUC) was not different in C and MS rats. AUC due to chronic restraint stress was not different in C and MS rats as well. Separate groups of rats were fed either a high salt diet (HSD, 4%NaCl), infused with AngII (200 ng/kg/min) or infused with AngII + HSD for one week. MAP was not different in C and MS rats fed a HSD (176±5 vs. 163±7 mmHg). AngII‐induced hypertension was not different in C and MS rats (146±4 vs. 150±1 mmHg). AngII+HSD increased MAP in MS rats compared to C rats (167±5 vs. 152±2 mmHg, respectively, ptime=&lt;0.05, pseparation= &lt;0.05 and pinteraction= &lt;0.05). We conclude that MS exacerbates AngII‐induced hypertension in the presence of HSD in DS rats.

Repeated Short-term (2h×14d) Emotional Stress Induces Lasting Depression-like Behavior in Mice

Chronic behavioral stress is a risk factor for depression. To understand chronic stress effects and the mechanism underlying stress-induced emotional changes, various animals model have been developed. We recently reported that mice treated with restraints for 2 h daily for 14 consecutive days (2h-14d or 2h×14d) show lasting depression-like behavior. Restraint provokes emotional stress in the body, but the nature of stress induced by restraints is presumably more complex than emotional stress. So a question remains unsolved whether a similar procedure with "emotional" stress is sufficient to cause depression-like behavior. To address this, we examined whether "emotional" constraints in mice treated for 2h×14d by enforcing them to individually stand on a small stepping platform placed in a water bucket with a quarter full of water, and the stress evoked by this procedure was termed "water-bucket stress". The water-bucket stress activated the hypothalamus-pituitary-adrenal gland (HPA) system in a manner similar to restraint as evidenced by elevation of serum glucocorticoids. After the 2h×14d water-bucket stress, mice showed behavioral changes that were attributed to depression-like behavior, which was stably detected >3 weeks after last water-bucket stress endorsement. Administration of the anti-depressant, imipramine, for 20 days from time after the last emotional constraint completely reversed the stress-induced depression-like behavior. These results suggest that emotional stress evokes for 2h×14d in mice stably induces depression-like behavior in mice, as does the 2h×14d restraint.

Chronic behavioral stress induces apical dendritic reorganization in pyramidal neurons of the medial prefrontal cortex

Both the hippocampus and the medial prefrontal cortex (mPFC) play an important role in the negative feedback regulation of hypothalamic–pituitary–adrenal (HPA) activity during physiologic and behavioral stress. Moreover, chronic behavioral stress is known to affect the morphology of CA3c pyramidal neurons in the rat, by reducing total branch number and length of apical dendrites. In the present study, we investigated the effects of behavioral stress on the mPFC, using the repeated restraint stress paradigm. Animals were perfused after 21 days of daily restraint, and intracellular iontophoretic injections of Lucifer Yellow were carried out in pyramidal neurons of layer II/III of the anterior cingulate cortex and prelimbic area. Cellular reconstructions were performed on apical and basal dendrites of pyramidal neurons in layer II/III of the anterior cingulate and prelimbic cortices. We observed a significant reduction on the total length (20%) and branch numbers (17%) of apical dendrites, and no significant reduction in basal dendrites. These cellular changes may impair the capacity of the mPFC to suppress the response of the HPA axis to stress, and offer an experimental model of stress-induced neocortical reorganization that may provide a structural basis for the cognitive impairments observed in post-traumatic stress disorder.