Immobilization stress elevates IP(3) receptor mRNA in adult rat hearts in a glucocorticoid-dependent manner

Gene expression of the type 1 and 2 inositol 1,4,5-trisphosphate (IP(3)) receptors in the rat cardiac atria and ventricles and their possible modulation by single immobilization stress was studied. Single immobilization stress significantly elevated mRNA levels for both types of these receptors. To evaluate the involvement of glucocorticoids in the modulation of the gene expression of IP(3) receptors by immobilization stress, we used adrenalectomized and/or hypophysectomized rats. Since adrenalectomy and/or hypophysectomy completely abolished increase in IP(3) receptor's mRNA levels after the immobilization, we conclude that immobilization stress elevates mRNA of type 1 and 2 IP(3) receptors, mainly through the glucocorticoid responsive element.     

Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs.Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4⁺CD127^?CD25^hi Tregs and CD4⁺CD127^?CD25⁺CD45RA⁺ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated.In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.     

Transient hypercapnic stress causes exaggerated and prolonged elevation of cardiac and renal interstitial norepinephrine levels in conscious hypertensive rats

The responses of sympathetic nerve activity to transient stress can be exaggerated in salt-sensitive (SS), hypertensive subjects. Cardiac and renal interstitial norepinephrine (iNE) levels during and after transient hypercapnia were investigated in conscious SS rats. Dahl SS and salt-resistant (SR) 6-wk-old rats were fed a high-salt diet, and at 12 wk iNE levels in the heart and kidney were determined using microdialysis with probes inserted in the left ventricular (LV) wall and kidney. A telemetry system determined blood pressure and heart rate (HR) in separate animals. After recovery from the operation, data were collected before, during, and after exposure to normoxic 10% CO(2) for 25 min under unanesthetized conditions. The plasma NE concentrations at baseline did not differ between the two strains. Both cardiac and renal iNE levels were much higher in SS rats than in SR rats at baseline as well as during hypercapnic stress. After stress, the markedly increased iNE levels of SS rats were prolonged in the LV as well as in the kidney. During hypercapnic stress, HR decreased in both SS and SR rats, while sudden increases in HR immediately after the withdrawal from stress were followed by its slower reduction in SS rats compared with SR rats. In conclusion, transient hypercapnic stress causes exaggerated and prolonged elevation of iNE levels in the heart as well as in kidneys of SS animals.     

Thyroid hormone regulation by stress and behavioral differences in adult male rats

Thyroid hormones are essential regulators of growth, development and normal bodily function and their release is coordinated by the hypothalamic-pituitary-thyroid (HPT) axis. While the HPT axis has been established as an acutely stress-responsive neuroendocrine system, relatively little is known about the mechanisms of its stress regulation. The present study examined acute stress-induced changes in peripheral hormone levels [triiodothyronine (T3); thyroxine (T4), thyroid-stimulating hormone (TSH), reverse triiodothyronine (rT3)] and central mRNA levels of regulators of the HPT axis [thyrotropin-releasing hormone (TRH), somatostatin (SST), type II deiodinase (D2)] in response to an inescapable tail-shock, a rodent model of stress. Additionally, we examined whether individual differences in spontaneous exploratory behavior in an open field test predicted basal levels of TH or differential susceptibility to the effects of stress. The stress condition was associated with decreases in peripheral T3, T4 and TSH, but not rT3, when compared with controls. No changes were observed in TRH or SST mRNA levels, but there was a trend suggesting stress-related increases in D2 mRNA. We also found that an animal's exploratory behavior in an unfamiliar open field arena was positively related to peripheral thyroid hormone levels and predicted the magnitude of stress-induced changes.In conclusion, we found suggestive evidence for stress-induced decrease in central drive HPT axis, but the central mechanisms of its stress regulation remain to be elucidated. Additionally, we found that individual differences in animals' exploratory behavior were correlated with peripheral TH levels.     

Dobutamine stress echocardiography in healthy adult male rats

Medical imaging market consists of several billion tests per year worldwide. Out of these, at least one third are cardiovascular procedures. Keeping in mind that each test represents a cost, often a risk, and a diagnostic hypothesis, we can agree that every unnecessary and unjustifiable test is one test too many. Small individual costs, risks, and wastes multiplied by billions of examinations per year represent an important population, society and environmental burden. Unfortunately, the appropriateness of cardiac imaging is extra-ordinarily low and there is little awareness in patients and physicians of differential costs, radiological doses, and long term risks of different imaging modalities. For a resting cardiac imaging test, being the average cost (not charges) of an echocardiogram equal to 1 (as a cost comparator), the cost of a CT is 3.1x, of a SPECT 3.27x, of a Cardiovascular Magnetic Resonance imaging 5.51x, of a PET 14.03x, and of a right and left heart catheterization 19.96x. For stress cardiac imaging, compared with the treadmill exercise test equal to 1 (as a cost comparator), the cost of stress echocardiography is 2.1x and of a stress SPECT scintigraphy is 5.7x. Biohazards and downstream long-term costs linked to radiation-induced oncogenesis should also be considered. The radiation exposure is absent in echo and magnetic resonance, and corresponds to 500 chest x rays for a sestamibi cardiac stress scan and to 1150 chest x rays for a thallium scan. The corresponding extra-risk in a lifetime of fatal cancer is 1 in 2000 exposed patients for a sestamibi stress and 1 in 1000 for a thallium scan. Increased awareness of economic, biologic, and environmental costs of cardiac imaging will hopefully lead to greater appropriateness, wisdom and prudence from both the prescriber and the practitioner. In this way, the sustainability of cardiac imaging will eventually improve.     

Oxidative stress impairs cardiac chemoreflexes in diabetic rats

We investigated the effects of diabetes mellitus and antioxidant treatment on the sensory and reflex function of cardiac chemosensory nerves in rats. Diabetes was induced by streptozotocin (STZ; 85 mg/kg ip). Subgroups of sham- and STZ-treated rats were chronically treated with an antioxidant, vitamin E (60 mg/kg per os daily, started 2 days before STZ). Animals were studied 6-8 wk after STZ injection. We measured renal sympathetic nerve activity (RSNA), mean arterial blood pressure (MABP), and cardiac vagal and sympathetic afferent activities in response to stimulation of chemosensitive sensory nerves in the heart by epicardial application of capsaicin (Caps) and bradykinin (BK). In cardiac sympathetic-denervated rats, Caps and BK (1-10.0 microg) evoked a vagal afferent mediated reflex depression of RSNA and MABP, which was significantly blunted in STZ-treated rats (P < 0.05). In vagal-denervated rats, Caps and BK (1-10.0 microg) evoked a sympathetic afferent-mediated reflex elevation of RSNA and MABP, which also was significantly blunted in STZ-treated rats (P < 0.05). Chronic vitamin E treatment effectively prevented these cardiac chemoreflex defects in STZ-treated rats without altering resting blood glucose or hemodynamics. STZ-treated rats with insulin replacement did not exhibit impaired cardiac chemoreflexes. In afferent studies, Caps and BK (0.1 g-10.0 microg) increased cardiac vagal and sympathetic afferent nerve activity in a dose-dependent manner in sham-treated rats. These responses were significantly blunted in STZ-treated rats. Vitamin E prevented the impairment of afferent discharge to chemical stimulation in STZ rats. The following were concluded: STZ-induced, insulin-dependent diabetes in rats extensively impairs the sensory and reflex properties of cardiac chemosensitive nerve endings, and these disturbances can be prevented by chronic treatment with vitamin E. These results suggest that oxidative stress plays an important role in the neuropathy of this autonomic reflex in diabetes.     

Salt stress affects glutamine synthetase activity and mRNA accumulation on potato plants in an organ-dependent manner

Ammonium assimilation into glutamine and glutamate is vital for plant growth as these are precursors for almost all nitrogenous compounds. Ammonium can be assimilated onto nitrogenous organic compounds by the concerted action of two enzymes that compose the glutamine synthetase (GS, EC 6.3.1.2) – glutamate synthase (Fd-GOGAT, EC 1.4.7.1; NADH–GOGAT, EC 1.4.1.14) cycle. Ammonium may also be directly incorporated into glutamate by the glutamate dehydrogenase (GDH, EC 1.4.1.2) aminating reaction. However, as GDH reversibly deaminates glutamate, its physiological role in vivo remains controversial. Potato has been classified as moderately tolerant to salinity. Potato GS is encoded by a small multigene family which is differentially regulated in an organ and age-dependent way. In this study, the effect of increasing concentrations of salinity in the soil in GS activity and gene-specific mRNA accumulation levels were studied on potato leaves and roots, as well as the biochemical parameters protein, chlorophyll, lipid peroxidation and proline levels, in order to evaluate the severity of the imposed stress. The data obtained suggests that when potato plants are subjected to salt stress, increased ammonium assimilation occurs in roots, due to an increased GS accumulation, along with a decreased assimilation in leaves. Regarding GS gene-specific mRNA accumulation, an organ-dependent response was also observed that contributes for the detected alteration in the ammonium assimilatory metabolism. This response may be a key feature for future genetic manipulations in order to increase crop productivity in salty soils. The possible contribution of GDH for ammonia assimilation was also investigated.     

Early life stress enhancement of limbic epileptogenesis in adult rats: mechanistic insights

Background

Exposure to early postnatal stress is known to hasten the progression of kindling epileptogenesis in adult rats. Despite the significance of this for understanding mesial temporal lobe epilepsy (MTLE) and its associated psychopathology, research findings regarding underlying mechanisms are sparse. Of several possibilities, one important candidate mechanism is early life ‘programming’ of the hypothalamic-pituitary-adrenal (HPA) axis by postnatal stress. Elevated corticosterone (CORT) in turn has consequences for neurogenesis and cell death relevant to epileptogenesis. Here we tested the hypotheses that MS would augment seizure-related corticosterone (CORT) release and enhance neuroplastic changes in the hippocampus.

Methodology/Principal Findings

Eight-week old Wistar rats, previously exposed on postnatal days 2–14 to either maternal separation stress (MS) or control brief early handling (EH), underwent rapid amygdala kindling. We measured seizure-induced serum CORT levels and post-kindling neurogenesis (using BrdU). Three weeks post-kindling, rats were euthanized for histology of the hippocampal CA3c region (pyramidal cell counts) and dentate gyrus (DG) (to count BrdU-labelled cells and measure mossy fibre sprouting). As in our previous studies, rats exposed to MS had accelerated kindling rates in adulthood. Female MS rats had heightened CORT responses during and after kindling (p<0.05), with a similar trend in males. In both sexes total CA3c pyramidal cell numbers were reduced in MS vs. EH rats post-kindling (p = 0.002). Dentate granule cell neurogenesis in female rats was significantly increased post-kindling in MS vs. EH rats.

Conclusions/Significance

These data demonstrate that early life stress results in enduring enhancement of HPA axis responses to limbic seizures, with increased hippocampal CA3c cell loss and augmented neurogenesis, in a sex-dependent pattern. This implicates important candidate mechanisms through which early life stress may promote vulnerability to limbic epileptogenesis in rats as well as to human MTLE and its associated psychiatric disorders.     

Cold-induced increases in phenylethanolamine N-methyltransferase (PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland

Previously, we reported that cold stress induces a rapid increase in adrenomedullary PNMT mRNA levels, followed by concomitant increases in PNMT immunoreactivity (10). In the present study, the extracellular signals mediating this adaptive response to stress were investigated using northern analysis and RNA slot-blot hybridization. Although adrenal denervation significantly diminished cold-induced increments in adrenomedullary PNMT mRNA levels, it did not completely abolish the cold stress response. In contrast to these results, splanchnectomy completely inhibited cold-induced increments in TH mRNAs in the same tissue samples. These findings indicate that the effects of cold exposure on PNMT mRNA levels are mediated by both neural and non-neural mechanisms, and that adrenal PNMT and TH are differentially regulated in response to cold stress. Surprisingly, the neural component of the PNMT stress response could not be attenuated by peripheral administration of chlorisondamine, a powerful nicotinic ganglionic blocking agent. In contrast, chlorisondamine was effective in inhibiting sympathetic neural activity, as judged by the drug's ability to completely block increases in blood pressure, heart rate, and plasma catecholamines resulting from spinal cord stimulation in pithed rats. The administration of atropine, a muscarinic receptor antagonist, also failed to inhibit cold-induced alterations in adrenal PNMT mRNA. These results suggest that the trans-synaptic induction of adrenal PNMT mRNA involves a non-cholinergic component, and that cold-induced increases in PNMT mRNA are not coupled to acetylcholine-mediated adrenal catecholamine release.     

Impact of brief oxidant stress on primary adult cardiac fibroblasts

Reperfusion of ischemic myocardium (I/R) is associated with local release of a brief pulse of reactive oxygen species. The purpose of this study was to determine the effects of brief H2O2 stimulation on primary adult cardiac fibroblast phenotype. We demonstrate that brief H2O2 exposure results in transient phosphorylations of p38 and ERK which peaked by 15 min. Proliferation was minimally affected by either H2O2 or MAPK inhibition. Pretreatment with SB203580 or U0126 revealed that p38 enhances or maintains migration rates while ERK retarded migration. Peroxide exposure increased necrosis from 4% at baseline to >12% while reducing apoptosis by 3.5-fold. p38 inhibition resulted in increased necrosis and apoptosis while ERK inhibition had minimal effects. In conclusion, primary adult cardiac fibroblasts exposed to brief H2O2 exhibit an altered phenotype characterized by reduced migration and apoptosis and increased necrosis resulting, in part, from the differential effects of p38 and ERK signaling.     

Agiotensin II induced cardiac hypertrophy in vivo is inhibited by cyclosporin A in adult rats

Recently, the calciumcalmodulindependent calcineurin pathway has been defined as a central pathway for the induction of cardiac hypertrophy. The purpose of this study was to determine if cardiac hypertrophy in animals chronically treated with angiotensin II (AngII), could be prevented by blocking this pathway with cyclosporin A (CsA). Female Wistar rats were treated with AngII by subcutaneous infusion and injected twice a day with CsA (25 mg/kg) for 7 days. In the AngII treated group there was a 30% increase in the heart/body weight ratio (p < 0.05 vs. control). The increase in heart weight was blocked with CsA. Substantial increases in ANF and MHC gene expression were detected in the AngII treated animals, which were either attenuated or blocked with CsA treatment. Thus, this study demonstrates that CsA does prevent the development of cardiac hypertrophy in Ang II treated rats, suggesting that the calciumcalmodulindependent calcineurin pathway is associated with angiotensin II induced hypertrophy in vivo.     

Effects of swim stress on mRNA and protein levels of steroid 5alpha-reductase isozymes in prefrontal cortex of adult male rats

The metabolite of progesterone, allopregnanolone, is among the most potent known ligands of the gamma-aminobutyric acid receptor complex (GABA(A)-R) in the central nervous system. This neuroactive steroid is markedly increased in an animal model of acute stress. Allopregnanolone is synthesized from progesterone by steroidogenic enzymes 5alpha-reductase (5alpha-R) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), with the former being the rate-limiting enzyme in this reaction sequence. In this paper, a quantitative RT-PCR method coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE) and Western blot were used to measure both mRNA and protein levels of 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2) isozymes in prefrontal cortex of male rats after acute swim stress situations. Our results demonstrate that both 5alpha-R isozymes are significantly higher in prefrontal cortex of male rats after acute swim stress in comparison with control rats. These data may open up a new research line that could improve our understanding of the role of 5alpha-R isozymes in processes that accompany stress situations.     

Influence of simulated ischemia on apoptosis induction by oxidative stress in adult cardiomyocytes of rats

Oxidative stress may cause apoptosis of cardiomyocytes in ischemic-reperfused myocardium. We investigated whether ischemia-reperfusion modifies the susceptibility of cardiomyocyte induction of apoptosis by oxidative stress. Ischemia was simulated by incubating isolated cardiomyocytes from adult rats in an anoxic, glucose-free medium, pH 6.4, for 3 h. Annexin V-fluorescein isothiocyanate/propidium iodide staining and the detection of DNA laddering were used as apoptotic markers. H(2)O(2) (7.5 micromol/l) induced apoptosis in 20.1 +/- 1.8% of cells under normoxic conditions but only 14.4 +/- 1.6% (n = 6, P < 0.05) after ischemia-reoxygenation. This partial protection of ischemic-reoxygenated cells was observed despite a reduction in their cellular glutathione content, from 11.4 +/- 1.9 in normoxic controls to 2.9 +/- 0.8 nmol/mg protein (n = 3, P < 0.05). Elevation of end-ischemic glutathione contents by pretreatment with 1 mmol/l N-acetylcysteine entirely protected ischemic-reoxygenated cells against induction of apoptosis by H(2)O(2). In conclusion, ischemia-reperfusion can protect cardiomyocytes against induction of apoptosis by exogenous oxidative stress. This endogenous protective effect is most clearly demonstrated when control and postischemic cardiomyocytes are compared at similar glutathione levels.     

Oxidative stress mediates cardiac fibrosis by enhancing transforming growth factor-beta1 in hypertensive rats

The methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism has been shown to be associated with cardiovascular disease and in patients with end-stage renal disease (ESRD). However, the relationship between MTHFR polymorphisms and cardiovascular disease (CVD) in patients on hemodialysis has not been examined. The aim of this study was to assess the association of polymorphisms of MTHFR gene with homocysteine (Hcy) and intimal medial thickness (IMT) in patients on hemodialysis. We performed case-control study involving107 patients with ESRD and 103 healthy controls. Plasma Hcy was measured in all the subjects and these subjects were genotyped for three MTHFR polymorphisms (C677T, A1298C, and G1793A). We observed significantly higher Hcy levels in patients as compared to controls. The frequency of MTHFR 1298CC genotype was significantly higher in ESRD patients than in controls (21.4% vs. 2.9%); the frequency of the MTHFR C677T genotypes did not differ between groups (26.1% vs. 17.4%). Compound heterozygous MTHFR 677CT/1298AC genotypes showed maximum association with the risk of ESRD (OR: 12.8; 5%CI: 1.64–10.01, P < 0.05). Concurrent occurrence of MTHFR 677CC wild genotype with either 1298CC or 1793GA significantly increased the risk of disease (OR: 7.20; 95%CI: 2.06–2.51, P < 0.001 and OR: 7.60; 95%CI: 1.68–34.35; P < 0.05, respectively). MTHFR 1298CC genotype was associated with higher Hcy levels. IMT was also significantly higher in patients with the 1298CC genotype (P < 0.05). Thus, A1298C polymorphism of MTHFR gene appears to be associated with the severity of carotid atherosclerosis and co-occurrence of MTHFR polymorphisms may be a risk factor for CVD in patients on hemodialysis.     

Effects of caffeic acid phenethyl ester on endotoxin-induced cardiac stress in rats: a possible mechanism of protection

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including myocardial dysfunction. The present study aimed to investigate the mechanism of caffeic acid phenethyl ester (CAPE) protection against LPS-induced cardiac stress. Rats were allocated into three groups; group 1 served as a normal control group, group 2 (LPS) received a single intraperitoneal injection of LPS (10 mg/kg), group 3 (LPS + CAPE) was injected intraperitoneally with CAPE (10 mg/kg/day; solubilized in saline containing 20% tween 20) throughout a period of 10 days prior to LPS injection. Rats were maintained 4 h before sacrifice. Caffeic acid phenethyl ester pretreatment normalized LPS-enhanced activities of serum creatine kinase (CK) and lactate dehydrogenase (LDH) as well as glutathione peroxidase (GPx), and myeloperoxidase (MPO) in cardiac tissue. A significant reduction of the elevated levels of serum tumor necrosis factor-alpha (TNF-α) as well as serum and cardiac nitrite/nitrate (NOx) ) was achieved after CAPE pretreatment. CAPE also restored malondialdelyde (MDA), reduced glutathione (GSH), and cytosolic calcium (Ca2+ ) levels in the heart. A marked induction of cardiac heme oxygenase-1 (HO-1) protein level was detected in CAPE-pretreated group. Whereas, LPS-induced reduction of adenosine triphosphate (ATP) and phosphocreatine (PCr) levels was insignificantly changed. Conclusively, the early treatment with CAPE maintained antioxidant defences, reduced oxidative injury, cytokine damage, and inflammation but did not markedly improve energy status in cardiac tissue. The beneficial effect of CAPE might be mediated, at least in part, by the superinduction of HO-1.