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.

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.

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.

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.

Noninvasive assessment of vascular structure and function in conscious rats based on in vivo imaging of the albino iris.

Experimental techniques allowing longitudinal studies of vascular disease progression or treatment effects are not readily available for most animal models. Thus, most existing studies are destined to either study individual time points or use large cohorts of animals. Here we describe a noninvasive technique for studying vascular disease that is based on in vivo imaging of the long posterior ciliary artery (LPCA) in the iris of albino rats. Using a slit-lamp biomicroscope, images of the LPCA were taken weekly in conscious normotensive Wistar Kyoto rats (WKY, n = 10) and spontaneously hypertensive rats (SHR, n = 10) for 10 wk. Using imaging software, we found that lumen diameter was significantly smaller and the wall-to-lumen (W/L) ratio larger in SHR than in WKY. Wall thickness was not different. Blood pressure correlated with the W/L ratio. Histology of the abdominal aorta also revealed a smaller lumen diameter and greater W/L ratio in SHR compared with WKY. Corneal application of the muscarinic receptor agonist pilocarpine elicited a dose-dependent vasodilation of the LPCA that could be antagonized by inhibition of nitric oxide synthase, suggesting that the pilocarpine response is mainly mediated by endothelium-derived nitric oxide. Consistent with endothelial dysfunction in SHR, pilocarpine-induced vasodilation was greater in WKY rats than in SHR. These findings indicate that in vivo imaging of the LPCA allows assessment of several structural and functional vascular parameters in conscious rats and that the LPCA responds to disease insults and pharmacologic treatments in a fashion that will make it a useful model for further studies.

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.

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.

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.

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.

Energy flux, more so than energy balance, protein intake, or fitness level, influences insulin-like growth factor-I system responses during 7 days of increased physical activity.

The purpose of this study was to determine the impact of dietary factors and exercise-associated factors on the response of IGF-I and its binding proteins (IGFBPs) during a period of increased physical activity. Twenty-nine men completed a 4-day (days 1-4) baseline period of a controlled energy balanced diet while maintaining their normal physical activity level followed by 7 days (days 5-11) of a 1,000 kcal/day increase in physical activity above their normal activity levels. Two subject groups, one sedentary (Sed, mean Vo(2peak): 39 mlxkg(-1)xmin(-1), n = 7) and one fit (FIT1, mean Vo(2peak): 56 ml.kg(-1)xmin(-1), n = 8) increased energy intake to maintain energy balance throughout the 7-day intervention. In two other fit subject groups (FIT2, n = 7 and FIT3, n = 7), energy intake remained at baseline resulting in a 1,000 kcal/day exercise-induced energy deficit. Of these, FIT2 received an adequate protein diet (0.9 g/kg), and FIT3 received a high-protein diet (1.8 g/kg). For all four groups, IGF-I, IGFBP-3, and the acid labile subunit (ALS) were significantly decreased by day 11 (27 +/- 4%, 10 +/- 2%, and 19 +/- 4%, respectively) and IGFBP-2 significantly increased by 49 +/- 21% following day 3. IGFBP-1 significantly increased only in the two negative energy balance groups, FIT2 (38 +/- 6%) and FIT3 (46 +/- 8%). Differences in initial fitness level and dietary protein intake did not alter the IGF-I system response to an acute increase in physical activity. Decreases in IGF-I were observed during a moderate increase in physical activity despite maintaining energy balance, suggesting that currently unexplained exercise-associated mechanisms, such as increased energy flux, regulate IGF-I independent of energy deficit.

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.

Altered secretion of growth hormone and luteinizing hormone after 84 h of sustained physical exertion superimposed on caloric and sleep restriction.

The pulsatile release of growth hormone (GH) and luteinizing hormone (LH) from the anterior pituitary gland is integral for signaling secretion of insulin-like growth factor (IGF)-I and testosterone, respectively. This study examined the hypothesis that 84 h of sustained physical exertion with caloric and sleep restriction alters the secretion of GH and LH. Ten male soldiers [22 yr (SD 3), 183 cm (SD 7), 87 kg (SD 8)] had blood drawn overnight from 1800 to 0600 every 20 min for GH, LH, and leptin and every 2 h for IGF-I (total and free), IGF binding proteins-1 and -3, testosterone (total and free), glucose, and free fatty acids during a control week and after 84 h of military operational stress. Time-series cluster and deconvolution analyses assessed the secretion parameters of GH and LH. Significant results (P < or = 0.05) were as follows: body mass (-3%), fat-free mass (-2.3%), and fat mass (-7.3%) declined after military operational stress. GH and LH secretion burst amplitude (approximately 50%) and overnight pulsatile secretion (approximately 50%), IGF binding protein-1 (+67%), and free fatty acids (+33%) increased, whereas leptin (-47%), total (-27%) and free IGF-I (-32%), total (-24%) and free testosterone (-30%), and IGF binding protein-3 (-6%) decreased. GH and LH pulse number were unaffected. Because GH and LH positively regulate IGF-I and testosterone, these data imply that the physiological strain induced a certain degree of peripheral resistance. During periods of energy deficiency, amplitude modulation of GH and LH pulses may precede alterations in pulse numbers.

Minimally invasive sampling of transdermal body fluid for the purpose of measuring insulin-like growth factor-I during exercise training.

Insulin-like growth factor-I (IGF-I) is a ubiquitous hormone that is secreted in both an endocrine and an autocrine/paracrine manner. IGF-I has conventionally been measured in serum; however, transdermal body fluid (TDF) remains as an unexplored biocompartment in which IGF-I also resides and may be more biologically relevant because of its proximity to tissues and cells. The purpose of this study was to compare IGF-I in serum versus IGF-I in TDF before and after 8 weeks of physical training. Twenty-eight healthy men (28 +/- 5 years old, 176 +/- 8 cm tall, weighing 83 +/- 11 kg) had TDF obtained by a novel, minimally invasive method that included the application of continuous vacuum pressure on forearm skin perforated with tiny micropores created by a focused beam from a laser system and also had blood obtained by venipuncture. An enzyme-linked immunosorbent assay measured total IGF-I concentrations. A repeated-measures analysis of variance (biocompartment x time) and Pearson Product Moment Correlation coefficients (P < or = 0.05) were used for statistical analyses. Data are presented as mean +/- SE. Total TDF IGF-I was significantly lower than serum IGF-I both before (TDF, 91 +/- 6 ng/mL; serum, 375 +/- 17 ng/mL) and after (TDF, 83 +/- 5 ng/mL; serum, 363 +/- 19 ng/mL) the exercise training. Serum and TDF IGF-I values were not significantly different pre- to post-training. Serum and TDF IGF-I levels were significantly correlated pre-training (r = 0.41), but not post-training (r = 0.34). The percent change between serum and TDF was not correlated (r = 0.09). This study has demonstrated that total IGF-I can be sampled and measured in TDF via a minimally invasive manner and is appreciably (approximately 76%) less than total IGF-I measured in serum. Additionally, the IGF-I measurements in these two biocompartments were not closely associated, possibly indicating an uncoupled, rather than a linked, regulation of IGF-I among the body's biocompartments.

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.

Effects of acute caloric restriction compared to caloric balance on the temporal response of the IGF-I system.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is a key regulator of metabolism during altered energy states. The IGF-I system components respond to prolonged caloric restriction but it is not clear if this system responds similarly to acute caloric restriction. The purpose of this study was to characterize the IGF-I system response to acute caloric restriction with a secondary purpose of determining if two isocaloric diets with different ratios of carbohydrate to fat alter the IGF-I system under conditions of caloric balance. MATERIALS/METHODS: A double-blind, placebo-controlled crossover design was used in which 27 subjects underwent three, 48-h experimental treatments: 1) caloric restriction 2) carbohydrate and 3) carbohydrate/fat. Blood was sampled periodically (6 time points total) for IGF-I (total and free), IGFBPs1-4, insulin and glucose. ANOVAs were used with significance set at P<0.05. RESULTS: Total IGF-I decreased 7% during CR (P=0.051) and remained stable during CHO and CHO/F. Free IGF-I decreased 43% during CR (P<0.05) and remained stable during CHO and CHO/F. IGFBP-1 increased by 445% during CR (P<0.05) compared to CHO and CHO/F with no changes for IGFBP-2, IGFBP-3 and IGFBP-4. There was no change in glucose or insulin during CR over the course of the study. Insulin and glucose increased (P<0.05) after a meal in both the CHO and CHO/F groups with no difference between these two groups. CONCLUSION: Our findings indicate that free IGF-I decreases and IGFBP-1 increases during caloric restriction, but they are not altered with diets differing in carbohydrate and fat content. Changes in free IGF-I and IGFBP-1 are sensitive to caloric restriction, and their measurement may be valuable in monitoring the physiological response to refeeding in those consuming suboptimal calories.

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.