Sterile inflammation induces vasculopathy and chronic lung injury in murine sickle cell disease.

Murine sickle cell disease (SCD) results in damage to multiple organs, likely mediated first by vasculopathy. While the mechanisms inducing vascular damage remain to be determined, nitric oxide bioavailability and sterile inflammation are both considered to play major roles in vasculopathy. Here, we investigate the effects of high mobility group box-1 (HMGB1), a pro-inflammatory damage-associated molecular pattern (DAMP) molecule on endothelial-dependent vasodilation and lung morphometrics, a structural index of damage in sickle (SS) mice. SS mice were treated with either phosphate-buffered saline (PBS), hE-HMGB1-BP, an hE dual-domain peptide that binds and removes HMGB1 from the circulation via the liver, 1-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(1H-imidazol-1-yl)phenyl]-1H-pyrrole-2-propanoic acid (N6022) or N-acetyl-lysyltyrosylcysteine amide (KYC) for three weeks. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant HMGB1 (r-HMGB1), which increases S-nitrosoglutathione reductase (GSNOR) expression by ∼80%, demonstrating a direct effect of HMGB1 to increase GSNOR. Treatment of SS mice with hE-HMGB1-BP reduced plasma HMGB1 in SS mice to control levels and reduced GSNOR expression in facialis arteries isolated from SS mice by ∼20%. These changes were associated with improved endothelial-dependent vasodilation. Treatment of SS mice with N6022 also improved vasodilation in SS mice suggesting that targeting GSNOR also improves vasodilation. SCD decreased protein nitrosothiols (SNOs) and radial alveolar counts (RAC) and increased GSNOR expression and mean linear intercepts (MLI) in lungs from SS mice. The marked changes in pulmonary morphometrics and GSNOR expression throughout the lung parenchyma in SS mice were improved by treating with either hE-HMGB1-BP or KYC. These data demonstrate that murine SCD induces vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism and suggest that HMGB1 and GSNOR might be effective therapeutic targets for reducing vasculopathy and chronic lung disease in humans with SCD.

Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability.

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1-2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

Astrocytic responses to high glucose impair barrier formation in cerebral microvessel endothelial cells.

Hyperglycemic conditions are prodromal to blood-brain barrier (BBB) impairment. The BBB comprises cerebral microvessel endothelial cells (CMECs) that are surrounded by astrocytic foot processes. Astrocytes express high levels of gap junction connexin 43 (Cx43), which play an important role in autocrine and paracrine signaling interactions that mediate gliovascular cross talk through secreted products. One of the key factors of the astrocytic "secretome" is vascular endothelial growth factor (VEGF), a potent angiogenic factor that can disrupt BBB integrity. We hypothesize that high-glucose conditions change the astrocytic expression of Cx43 and increase VEGF secretion leading to impairment of CMEC barrier properties in vitro and in vivo. Using coculture of neonatal rat astrocytes and CMEC, we mimic hyperglycemic conditions using high-glucose (HG) feeding media and show a significant decrease in Cx43 expression and the corresponding increase in secreted VEGF. This result was confirmed by the analyses of Cx43 and VEGF protein levels in the brain cortex samples from the type 2 diabetic rat (T2DN). To further characterize inducible changes in BBB, we measured transendothelial cell electrical resistance (TEER) and tight junction protein levels in cocultured conditioned astrocytes with isolated rat CMEC. The coculture monolayer's integrity and permeability were significantly compromised by HG media exposure, which was indicated by decreased TEER without a change in tight junction protein levels in CMEC. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross talk between astrocytes and CMEC, which could be one explanation for cerebral BBB disruption in diabetic conditions.

Cilia proteins are biomarkers of altered flow in the vasculature.

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.

Established, New and Emerging Concepts in Brain Vascular Development.

In this review, we discuss the state of our knowledge as it relates to embryonic brain vascular patterning in model systems zebrafish and mouse. We focus on the origins of endothelial cell and the distinguishing features of brain endothelial cells compared to non-brain endothelial cells, which is revealed by single cell RNA-sequencing methodologies. We also discuss the cross talk between brain endothelial cells and neural stem cells, and their effect on each other. In terms of mechanisms, we focus exclusively on Wnt signaling and the recent developments associated with this signaling network in brain vascular patterning, and the benefits and challenges associated with strategies for targeting the brain vasculature. We end the review with a discussion on the emerging areas of meningeal lymphatics, endothelial cilia biology and novel cerebrovascular structures identified in vertebrates.

Growth Hormone and Insulin-like Growth Factor-I Molecular Weight Isoform Responses to Resistance Exercise Are Sex-Dependent.

Purpose: To determine if acute resistance exercise-induced increases in growth hormone (GH) and insulin-like growth factor-I (IGF-I) were differentially responsive for one or more molecular weight (MW) isoforms and if these responses were sex-dependent. Methods: College-aged men (n = 10) and women (n = 10) performed an acute resistance exercise test (ARET; 6 sets, 10 repetition maximum (10-RM) squat, 2-min inter-set rest). Serum aliquots from blood drawn Pre-, Mid-, and Post-ARET (0, +15, and +30-min post) were processed using High Performance Liquid Chromatography (HPLC) fractionation and pooled into 3 MW fractions (Fr.A: >60; Fr.B: 30-60; Fr.C: <30 kDa). Results: We observed a hierarchy of serum protein collected among GH fractions across all time points independent of sex (Fr.C > Fr.A > Fr.B, p ≤ 0.03). Sex × time interactions indicated that women experienced earlier and augmented increases in all serum GH MW isoform fraction pools (p < 0.05); however, men demonstrated delayed and sustained GH elevations (p < 0.01) in all fractions through +30-min of recovery. Similarly, we observed a sex-independent hierarchy among IGF-I MW fraction pools (Fr.A > Fr.B > Fr.C, p ≤ 0.01). Furthermore, we observed increases in IGF-I Fr. A (ternary complexes) in men only (p ≤ 0.05), and increases in Fr.C (free/unbound IGF-I) in women only (p ≤ 0.05) vs. baseline, respectively. Conclusions: These data indicate that the processing of GH and IGF-I isoforms from the somatotrophs and hepatocytes are differential in their response to strenuous resistance exercise and reflect both temporal and sex-related differences.

Astrocytes influence medulloblastoma phenotypes and CD133 surface expression.

The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.

Effect of high glucose condition on glucose metabolism in primary astrocytes.

In the brain, glucose enters astrocytes through glucose transporter (GLUT1) and either enters glycolysis or the glycogen shunt. Astrocytes meet the energy needs of neurons by building up and breaking down their glycogen supply. High glucose exposure causes astrocyte dysregulation, but its effects on glucose metabolism are relatively unknown. We hypothesized that high glucose conditioning induces a glycogenic state in the astrocyte, resulting in an inefficient mobilization of substrates when challenged with glucose deprivation. Using neonatal rat astrocytes, we used normal glucose (NG, 5.5 mM) vs. high glucose (HG, 25 mM) feeding media and measured cell membrane GLUT1 expression, glucose analog uptake, glycogen content, and cellular bioenergetics. This study demonstrates that HG conditioning causes increased glucose analog uptake (p < 0.05) without affecting GLUT1 membrane expression when compared to NG conditioned astrocytes. Increased glucose uptake in HG astrocytes is associated with higher baseline glycogen content compared to NG exposed astrocytes (p < 0.05). When challenged with glucose deprivation, HG astrocytes break down more than double the amount of glycogen molecules compared to NG astrocytes, although they break down a similar percentage of the starting glycogen stores (NG = 62%, HG = 55%). Additionally, HG conditioning negatively impacts astrocyte maximal respiration and glycolytic reserve capacity assessed by the Seahorse mitochondrial stress test and glycolytic stress test, respectively (p < 0.05). These results suggest that HG conditioning shifts astrocytes towards glycogen storage at baseline. Despite increased glycogen storage, HG astrocytes demonstrate decreased metabolic efficiency and capacity putting them at higher risk during extended periods of glucose deprivation.

Interaction of tumor cells and astrocytes promotes breast cancer brain metastases through TGF-ß2/ANGPTL4 axes.

Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-ß2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-ß2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-ß2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.

High glucose conditioned neonatal astrocytes results in impaired mitogenic activity in cerebral microvessel endothelial cells in co-culture.

Angiogenesis is a highly complex and coordinated process in the brain. Under normal conditions, it is a vital process in growth and development, but under adverse conditions such as diabetes mellitus, it can lead to severe pathology. Astrocytes are a key constituent of the neurovascular unit and contribute to cerebral function, not only bridging the gap between metabolic supplies from blood vessels to neurons, but also regulating angiogenesis. Astrocytes affect angiogenesis by secreting angiogenic factors such as vascular endothelial growth factor (VEGF) into its microenvironment and regulating mitogenic activity in cerebral microvessel endothelial cells (CMEC). We hypothesized that astrocytes conditioned in high glucose media would produce and secrete decreased VEGF which would lead to impaired proliferation, migration, and tube formation of CMEC in vitro. Using neonatal rat astrocytes, we used normal glucose (NG, 5.5mM) vs. high glucose (HG, 25mM) feeding media and measured VEGF message and protein levels as well as secreted VEGF. We co-cultured conditioned astrocytes with isolated rat CMEC and measured mitogenic activity of endothelial cells using BrdU assay, scratch recovery assay, and tube formation assay. HG astrocytes produced and secreted decreased VEGF protein and resulted in impaired mitogenic activity when co-cultured with CMEC as demonstrated by decreased BrdU uptake, decreased scratch recovery, and slower tube formation. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross-talk between astrocytes and CMEC which could be one explanation for cerebral microangiopathy seen in diabetic conditions.

Characterization of growth hormone disulfide-linked molecular isoforms during post-exercise release vs nocturnal pulsatile release reveals similar milieu composition.

OBJECTIVE: To characterize the influence of mode (aerobic/resistance) and volume of exercise (moderate/high) on circulating GH immediately post-exercise as well as following the onset of sleep. DESIGN: This study used repeated measures in which subjects randomly completed 5 separate conditions: control (no exercise), moderate volume resistance exercise (MR), high-volume resistance exercise (HR), moderate volume aerobic exercise (MA), and high volume aerobic exercise (HA). METHODS: Subjects had two overnight stays on each of the 5 iterations. Serial blood draws began as soon as possible after the completion of the exercise session. Blood was obtained every 20 min for 24-h. GH was measured using a chemiluminescent immunoassay. Pooled samples representing post exercise (PE) and first nocturnal pulse (NP) were divided into two aliquots. One of these aliquots was chemically reduced by adding 10 mM glutathione (GSH) to break down disulfide-linked aggregates. RESULTS: No differences were observed when pooling GH response at post-exercise (2.02 ±â€¯0.21) and nocturnal pulse (2.63 ±â€¯0.51; p = .32). Pairwise comparisons revealed main effect differences between controls (1.19 ±â€¯0.29) and both MA (2.86 ±â€¯0.31; p = .009) and HA (3.73 ±â€¯0.71; p = .001). Both MA (p = .049) and HA (p = .035) responses were significantly larger than the MR stimulus (1.96 ±â€¯0.28). With GSH reduction, controls significantly differed from MA (p = .018) and HA (p = .003) during PE, but only differed from HA (p = .003) during NP. CONCLUSIONS: This study demonstrated similar GH responses to exercise and nocturnal pulse, indicating that mode and intensity of exercise does not proportionately affect GH dimeric isoform concentration.

Regulation of Cerebral Blood Flow: Response to Cytochrome P450 Lipid Metabolites.

There have been numerous reviews related to the cerebral circulation. Most of these reviews are similar in many ways. In the present review, we thought it important to provide an overview of function with specific attention to details of cerebral arterial control related to brain homeostasis, maintenance of neuronal energy demands, and a unique perspective related to the role of astrocytes. A coming review in this series will discuss cerebral vascular development and unique properties of the neonatal circulation and developing brain, thus, many aspects of development are missing here. Similarly, a review of the response of the brain and cerebral circulation to heat stress has recently appeared in this series (8). By trying to make this review unique, some obvious topics were not discussed in lieu of others, which are from recent and provocative research such as endothelium-derived hyperpolarizing factor, circadian regulation of proteins effecting cerebral blood flow, and unique properties of the neurovascular unit. © 2018 American Physiological Society. Compr Physiol 8:801-821, 2018.

Differential basal and exercise-induced IGF-I system responses to resistance vs. calisthenic-based military readiness training programs.

OBJECTIVE: The purpose of this study was to: 1) evaluate differential responses of the IGF-I system to either a calisthenic- or resistance exercise-based program and 2) determine if this chronic training altered the IGF-I system during an acute resistance exercise protocol. DESIGN: Thirty-two volunteers were randomly assigned into a resistance exercise-based training (RT) group (n=15, 27±5y, 174±6cm, 81±12kg) or a calisthenic-based training group (CT) (n=17, 29±5y, 179±8cm, 85±10kg) and all underwent 8weeks of exercise training (1.5h/d, 5d/wk). Basal blood was sampled pre- (Week 0), mid- (Week 4) and post-training (Week 8) and assayed for IGF-I system analytes. An acute resistance exercise protocol (AREP) was conducted preand post-training consisting of 6 sets of 10 repetitions in the squat with two minutes of rest in between sets and the IGF-I system analytes measured. A repeated measures ANOVA (p≤0.05) was used for statistical analysis. RESULTS: No interaction or within-subject effects were observed for basal total IGF-I, free IGF-I, or IGFBP-1. IGFBP-2 (pre; 578.6±295.7post-training; 14.3±1.9µg/mL; p=0.01). An interaction was observed for the RT group as IGFBP-3 increased from pre to mid (3462.4±216.4 vs. 3962.2±227.9ng/mL), but was not significant at the post-training time point (3770.3±228.7ng/mL). AREP caused all analytes except free IGF-I (40% decrease) to increase (17-27%; p=0.001) during exercise, returning to baseline concentration into recovery. CONCLUSION: Post-training, bioavailable IGF-I recovered more rapidly post-exercise. 8wks of chronic physical training resulted in increased basal IGFBP-2 and IGFBP-3, decreased ALS, increased pre-AREP free IGF-I and a more rapid free IGF-I recovery post-AREP. While total IGF-I was insensitive to chronic physical training, changes were observed with circulating IGFBPs and bioavailable IGF-I. To glean the most robust information on the effects of exercise training, studies must move beyond relying solely on total IGF-I measures and should consider IGFBPs and bioavailable IGF-I as these components of the circulating IGF-I system are essential determinants of IGF-I physiological action.

Expression of CYP 4A ω-hydroxylase and formation of 20-hydroxyeicosatetreanoic acid (20-HETE) in cultured rat brain astrocytes.

Astrocytes secrete vasodilator and vasoconstrictor factors via end feet processes, altering blood flow to meet neuronal metabolic demand. Compared to what is known about the ability of astrocytes to release factors that dilate local cerebral vasculature, very little is known regarding the source and identity of astrocyte derived constricting factors. The present study investigated if astrocytes express CYP 4A ω-hydroxylase and metabolize arachidonic acid (AA) to 20-hydroxyeicotetraenoic acid (20-HETE) that regulates KCa channel activity in astrocytes and cerebral arterial myocyte contractility. Here we report that cultured astrocytes express CYP 4A2/3 ω-hydroxylase mRNA and CYP 4A protein and produce 20-HETE and the CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) when incubated with AA. The production of 20-HETE and EETs was enhanced following stimulation of metabotropic glutamate receptors (mGluR) on the astrocytes. Exogenous application of 20-HETE attenuated, whereas inhibition of 20-HETE production with HET-0016 increased the open state probabilities (NPo) of 71pS and 161pS KCa single-channel currents recorded from astrocytes. Exposure of isolated cerebral arterial myocytes to conditioned media from cultured astrocytes caused shortening of the length of freshly isolated cerebral arterial myocytes that was not evident following inhibition of astrocyte 20-HETE synthesis and action. These findings suggest that astrocytes not only release vasodilator EETs in response to mGluR stimulation but also synthetize and release the cerebral arterial myocyte constrictor 20-HETE that also functions as an endogenous inhibitor of the activity of two types of KCa channel currents found in astrocytes.

Elevated Aminopeptidase P Attenuates Cerebral Arterial Responses to Bradykinin in Fawn-Hooded Hypertensive Rats.

Cerebral arterial myogenic and autoregulatory responses are impaired in Fawn Hooded hypertensive (FHH) rats. Cerebral autoregulatory responses are restored in the congenic rat strain in which a segment of chromosome 1 from the Brown Norway (BN) rat was transferred into the FHH genetic background (FHH.1BN). The impact of this region on cerebral arterial dilator responses remains unknown. Aminopeptidase is a gene that was transferred into the FHH genetic background to generate the FHH.1BN rats and is responsible for degradation of the vasodilator bradykinin. Thus, we hypothesized that FHH rats will have increased aminopeptidase P levels with impaired cerebral arterial responses to bradykinin compared to BN and FHH.1BN rats. We demonstrated higher cerebral arterial expression of aminopeptidase P in FHH compared to BN rats. Accordingly, we demonstrated markedly impaired cerebral arterial dilation to bradykinin in FHH compared to BN rats. Interestingly, aminopeptidase P expression was lower in FHH.1BN compared to FHH rats. Decreased aminopeptidase P levels in FHH.1BN rats were associated with increased cerebral arterial bradykinin-induced dilator responses. Aminopeptidase P inhibition by apstatin improved cerebral arterial bradykinin dilator responses in FHH rats to a level similar to FHH.1BN rats. Unlike bradykinin, cerebral arterial responses to acetylcholine were similar between FHH and FHH.1BN groups. These findings indicate decreased bradykinin bioavailability contributes to impaired cerebral arterial dilation in FHH rats. Overall, these data indicate an important role of aminopeptidase P in the impaired cerebral arterial function in FHH rat.

Twenty-hour growth hormone secretory profiles after aerobic and resistance exercise.

INTRODUCTION: The pulsatile secretion pattern of growth hormone (GH) is an important parameter of GH action at peripheral tissues, and more information is needed on how exercise impacts GH secretion. This study hypothesized that both aerobic and resistance exercise would exhibit dose-response relationships with respect to exercise duration and 20-h postexercise GH secretion. METHODS: Eight healthy men randomly completed five separate conditions: 1) control (no exercise; CON), 2) a moderate-duration (1-h) aerobic exercise session (MA), 3) a long-duration (2-h) aerobic exercise session (LA), 4) a moderate-duration (1-h) resistance exercise session (MR), and 5) a long-duration (2-h) resistance exercise session (LR). Exercise intensity, diet, sleep, and physical activity were strictly controlled during each condition, and blood was sampled postexercise every 20 min for 20 h, and GH secretion parameters were analyzed via cluster and deconvolution analyses. RESULTS: Only the 2-h aerobic exercise bout resulted in a significant amplification of GH secretion as evidenced by increases in GH burst peak amplitude (∼100%), basal GH secretion rate (∼127%), total GH basal secretion (∼120%), total pulsatile secretion (∼88%), and total GH secretion (∼89%) over the control (i.e., no exercise) condition. GH secretions for the resistance exercise conditions were not different from control. CONCLUSIONS: The fact that the 2-h aerobic exercise condition resulted in higher energy expenditure than the other exercise conditions could offer a partial explanation for the greater GH amplification because of the metabolic effects that GH exerts in stimulating postexercise lipolysis. We conclude that extending the duration of aerobic exercise, but not resistance exercise, from 1- to 2-h significantly amplifies GH secretion during a 20-h period.

Epoxyeicosatrienoic acids pretreatment improves amyloid ß-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes.

Amyloid-ß (Aß) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aß accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aß-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aß (1, 10 µM) exposure versus absence of Aß. MS-PPOH preincubation also aggravated Aß-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aß. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aß-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aß toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aß reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aß inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

Astrocytes directly influence tumor cell invasion and metastasis in vivo.

Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.