Vascular effects of disrupting endothelial mTORC1 signaling in obesity.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling complex is emerging as a critical regulator of cardiovascular function with alterations in this pathway implicated in cardiovascular diseases. In this study, we used animal models and human tissues to examine the role of vascular mTORC1 signaling in the endothelial dysfunction associated with obesity. In mice, obesity induced by high-fat/high-sucrose diet feeding for ∼2 mo resulted in aortic endothelial dysfunction without appreciable changes in vascular mTORC1 signaling. On the other hand, chronic high-fat diet feeding (45% or 60% kcal: ∼9 mo) in mice resulted in endothelial dysfunction associated with elevated vascular mTORC1 signaling. Endothelial cells and visceral adipose vessels isolated from obese humans display a trend toward elevated mTORC1 signaling. Surprisingly, genetic disruption of endothelial mTORC1 signaling through constitutive or tamoxifen inducible deletion of endothelial Raptor (critical subunit of mTORC1) did not prevent or rescue the endothelial dysfunction associated with high-fat diet feeding in mice. Endothelial mTORC1 deficiency also failed to reverse the endothelial dysfunction evoked by a high-fat/high-sucrose diet in mice. Taken together, these data show increased vascular mTORC1 signaling in obesity, but this vascular mTORC1 activation appears not to be required for the development of endothelial impairment in obesity.

Muscle contraction induced arterial shear stress increases endothelial nitric oxide synthase phosphorylation in humans.

We determined if local increases in brachial artery shear during repetitive muscle contractions induce changes in protein expression of endothelial nitric oxide synthase (eNOS) and/or phosphorylated (p-)eNOS at Ser1177, the primary activation site on eNOS, in endothelial cells (ECs) of humans. Seven young male subjects (25 ± 1 yr) performed 20 separate bouts (3 min each) of rhythmic forearm exercise at 20% of maximum over a 2-h period. Each bout of exercise was separated by 3 min of rest. An additional six male subjects (24 ± 1 yr) served as time controls (no exercise). ECs were freshly isolated from the brachial artery using sterile J-wires through an arterial catheter at baseline and again after the 2-h exercise or time control period. Expression of eNOS or p-eNOS Ser1177 in ECs was determined via immunofluorescence. Brachial artery mean shear rate was elevated compared with baseline and the time control group throughout the 2-h exercise protocol (P < 0.001). p-eNOS Ser1177 expression was increased 57% in ECs in the exercise group [0.06 ± 0.01 vs. 0.10 ± 0.02 arbitrary units (au), P = 0.02] but not in the time control group (0.08 ± 0.01 vs. 0.07 ± 0.01 au, P = 0.72). In contrast, total eNOS expression did not change in either the exercise (0.13 ± 0.04 vs. 0.12 ± 0.03 au) or time control (0.12 ± 0.03 vs. 0.11 ± 0.03 au) group (P > 0.05 for both). Our novel results suggest that elevations in brachial artery shear increase eNOS Ser1177 phosphorylation in the absence of changes in total eNOS in ECs of young healthy male subjects, suggesting that this model is sufficient to alter posttranslational modification of eNOS activity in vivo in humans.NEW & NOTEWORTHY Elevations in brachial artery shear in response to forearm exercise increased endothelial nitric oxide synthase Ser1177 phosphorylation in brachial artery endothelial cells of healthy humans. Our present study provides the first evidence in humans that muscle contraction-induced increases in conduit arterial shear lead to in vivo posttranslational modification of endothelial nitric oxide synthase activity in endothelial cells.

Myeloperoxidase is increased in human cerebral aneurysms and increases formation and rupture of cerebral aneurysms in mice.

BACKGROUND AND PURPOSE: Cerebral aneurysm (CA) affects 3% of the population and is associated with hemodynamic stress and inflammation. Myeloperoxidase, a major oxidative enzyme associated with inflammation, is increased in patients with CA, but whether myeloperoxidase contributes to CA is not known. We tested the hypotheses that myeloperoxidase is increased within human CA and is critical for formation and rupture of CA in mice. METHODS: Blood was drawn from the lumen of CAs and femoral arteries of 25 patients who underwent endovascular coiling of CA, and plasma myeloperoxidase concentrations were measured with ELISA. Effects of endogenous myeloperoxidase on CA formation and rupture were studied in myeloperoxidase knockout mice and wild-type (WT) mice using an angiotensin II-elastase induction model of CA. In addition, effects of myeloperoxidase on inflammatory gene expression in endothelial cells were analyzed. RESULTS: Plasma concentrations of myeloperoxidase were 2.7-fold higher within CA than in femoral arterial blood in patients with CA. myeloperoxidase-positive cells were increased in aneurysm tissue compared with superficial temporal artery of patients with CA. Incidence of aneurysms and subarachnoid hemorrhage was significantly lower in myeloperoxidase knockout than in WT mice. In cerebral arteries, proinflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2 (COX2), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C motif) ligand (XCL1), matrix metalloproteinase (MMP) 8, cluster of differentiation 68 (CD68), and matrix metalloproteinase 13, and leukocytes were increased, and α-smooth muscle actin was decreased, in WT but not in myeloperoxidase knockout mice after induction of CA. Myeloperoxidase per se increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells. CONCLUSIONS: These findings suggest that myeloperoxidase may contribute importantly to formation and rupture of CA.

Estrogen-mediated individual differences in female rat voluntary running behavior.

Regular exercise has numerous health benefits, but the human population displays significant variability in exercise participation. Rodent models, such as voluntary wheel running (VWR) in rats, can provide insight into the underlying mechanisms of exercise behavior and its regulation. In this study, we focused on the role of estrogen on VWR in female rats. Female rats run more than males, and we aimed to determine to what extent running levels in females were regulated by estrogen signaling. The running behavior of rats (duration, speed, and total distance run) was measured under normal physiological conditions, ovariectomy (OVX), and estrogen replacement in an OVX background. Results show cyclic variations in running linked to the estrous cycle. Ovariectomy markedly reduced running and eliminated the cyclic pattern. Estrogen replacement through estradiol benzoate (EB) injections and osmotic minipumps reinstated running activity to pre-OVX levels and restored the cyclic pattern. Importantly, individual differences and ranking are preserved such that high versus low runners before OVX remain high and low runners after treatment. Further analysis revealed that individual variation in running distance was primarily caused by rats running different speeds, but rats also varied in running duration. However, it is noteworthy that this model also displays features distinct from estrogen-driven running behavior under physiological conditions, notably a delayed onset and a broader duration of running activity. Collectively, this estrogen causality VWR model presents a unique opportunity to investigate sex-specific mechanisms that control voluntary physical activity.NEW & NOTEWORTHY This study investigates estrogen's role in voluntary wheel running (VWR) behavior in female rats. Female rats exhibit greater running than males, with estrogen signaling regulating this activity. The estrous cycle influences running, whereas ovariectomy reduces it, and estrogen replacement restores it, maintaining individual differences under all conditions. Both running speed and duration contribute to VWR variations. These findings emphasize individual estrogen regulation in female exercise and provide an estrogen replacement animal model for investigating neurobiological underpinnings that drive voluntary exercise behavior.

Subcellular partitioning of protein kinase activity revealed by functional kinome profiling.

Protein kinases and their substrates form signaling networks partitioned across subcellular compartments to facilitate critical biological processes. While the subcellular roles of many individual kinases have been elucidated, a comprehensive assessment of the synaptic subkinome is lacking. Further, most studies of kinases focus on transcript, protein, and/or phospho-protein expression levels, providing an indirect measure of protein kinase activity. Prior work suggests that gene expression levels are not a good predictor of protein function. Thus, we assessed global serine/threonine protein kinase activity profiles in synaptosomal, nuclear, and cytosolic fractions from rat frontal cortex homogenate using peptide arrays. Comparisons made between fractions demonstrated differences in overall protein kinase activity. Upstream kinase analysis revealed a list of cognate kinases that were enriched in the synaptosomal fraction compared to the nuclear fraction. We identified many kinases in the synaptic fraction previously implicated in this compartment, while also identifying other kinases with little or no evidence for synaptic localization. Our results show the feasibility of assessing subcellular fractions with peptide activity arrays, as well as suggesting compartment specific activity profiles associated with established and novel kinases.

Determination of Diffusion Kinetics of Ketamine in Brain Tissue: Implications for in vitro Mechanistic Studies of Drug Actions.

Ketamine has been in use for over 50 years as a general anesthetic, acting primarily through blockade of N-methyl-D-aspartate receptors in the brain. Recent studies have demonstrated that ketamine also acts as a potent and rapid-acting antidepressant when administered at sub-anesthetic doses. However, the precise mechanism behind this effect remains unclear. We examined the diffusion properties of ketamine in brain tissue to determine their effects in in vitro studies related to the antidepressant action of ketamine. Brain slices from adult mice were exposed to artificial cerebrospinal fluid (aCSF) containing ∼17 µM ketamine HCl for varying amounts of time. The amount of ketamine within each slice was then measured by tandem high-performance liquid chromatography - mass spectrometry to characterize the diffusion of ketamine into brain tissue over time. We successfully modeled the diffusion of ketamine into brain tissue using a mono-exponential function with a time constant of τ = 6.59 min. This curve was then compared to a one-dimensional model of diffusion yielding a diffusion coefficient of approximately 0.12 cm2⋅s-1 for ketamine diffusing into brain tissue. The brain:aCSF partition coefficient for ketamine was determined to be approximately 2.76. Our results suggest that the diffusion properties of ketamine have a significant effect on drug concentrations achieved within brain tissue during in vitro experiments. This information is vital to determine the ketamine concentration necessary for in vitro slice preparation to accurately reflect in vivo doses responsible for its antidepressant actions.

Corticosterone as a Potential Confounding Factor in Delineating Mechanisms Underlying Ketamine's Rapid Antidepressant Actions.

Recent research into the rapid antidepressant effect of subanesthetic doses of ketamine have identified a series of relevant protein cascades activated within hours of administration. Prior to, or concurrent with, these activation cascades, ketamine treatment generates dissociative and psychotomimetic side effects along with an increase in circulating glucocorticoids. In rats, we observed an over 3-fold increase in corticosterone levels in both serum and brain tissue, within an hour of administration of low dose ketamine (10 mg/kg), but not with (2R, 6R)-hydroxynorketamine (HNK) (10 mg/kg), a ketamine metabolite shown to produce antidepressant-like action in rodents without inducing immediate side-effects. Hippocampal tissue from ketamine, but not HNK, injected animals displayed a significant increase in the expression of sgk1, a downstream effector of glucocorticoid receptor signaling. To examine the role conscious sensation of ketamine's side effects plays in the release of corticosterone, we assessed serum corticosterone levels after ketamine administration while under isoflurane anesthesia. Under anesthesia, ketamine failed to increase circulating corticosterone levels relative to saline controls. Concurrent with its antidepressant effects, ketamine generates a release of glucocorticoids potentially linked to disturbing cognitive side effects and the activation of distinct molecular pathways which should be considered when attempting to delineate the molecular mechanisms of its antidepressant function.

Novel role for endogenous hepatocyte growth factor in the pathogenesis of intracranial aneurysms.

Inflammation plays a key role in formation and rupture of intracranial aneurysms. Because hepatocyte growth factor (HGF) protects against vascular inflammation, we sought to assess the role of endogenous HGF in the pathogenesis of intracranial aneurysms. Circulating HGF concentrations in blood samples drawn from the lumen of human intracranial aneurysms or femoral arteries were compared in 16 patients. Tissue from superficial temporal arteries and ruptured or unruptured intracranial aneurysms collected from patients undergoing clipping (n=10) were immunostained with antibodies to HGF and its receptor c-Met. Intracranial aneurysms were induced in mice treated with PF-04217903 (a c-Met antagonist) or vehicle. Expression of inflammatory molecules was also measured in cultured human endothelial, smooth muscle cells and monocytes treated with lipopolysaccharides in presence or absence of HGF and PF-04217903. We found that HGF concentrations were significantly higher in blood collected from human intracranial aneurysms (1076±656 pg/mL) than in femoral arteries (196±436 pg/mL; P<0.001). HGF and c-Met were detected by immunostaining in superficial temporal arteries and in both ruptured and unruptured human intracranial aneurysms. A c-Met antagonist did not alter the formation of intracranial aneurysms (P>0.05), but significantly increased the prevalence of subarachnoid hemorrhage and decreased survival in mice (P<0.05). HGF attenuated expression of vascular cell adhesion molecule-1 (P<0.05) and E-Selectin (P<0.05) in human aortic endothelial cells. In conclusion, plasma HGF concentrations are elevated in intracranial aneurysms. HGF and c-Met are expressed in superficial temporal arteries and in intracranial aneurysms. HGF signaling through c-Met may decrease inflammation in endothelial cells and protect against intracranial aneurysm rupture.

Retroviral-infection increases tumorigenic potential of MDA-MB-231 breast carcinoma cells by expanding an aldehyde dehydrogenase (ALDH1) positive stem-cell like population.

Retroviral transformation has been associated with pro-proliferative oncogenic signaling in human cells. The current study demonstrates that transduction of human breast carcinoma cells (MDA-MB231) with LXSN and QCXIP retroviral vectors causes significant increases in growth rate, clonogenic fraction, and aldehyde dehydrogenase-1 positive cells (ALDH1+), which is associated with increased steady-state levels of cancer stem cell populations. Furthermore, this retroviral-induced enhancement of cancer cell growth in vitro was also accompanied by a significant increase in xenograft tumor growth rate in vivo. The retroviral induced increases in cancer cell growth rate were partially inhibited by treatment with 100 U/ml polyethylene glycol-conjugated-(PEG)-superoxide dismutase and/or PEG-catalase. These results show that retroviral infection of MDA-MB231 human breast cancer cells is capable of enhancing cell proliferation and cancer stem cell populations as well as suggesting that modulation of reactive oxygen species-induced pro-survival signaling pathways may be involved in these effects.