Kevin R. Rarick

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.

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

Amyloid-β (Aβ) has long been implicated as a causative protein in Alzheimers disease. Cellular Aβ accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimers disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aβ-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 Aβ (1, 10 μM) exposure versus absence of Aβ. MS-PPOH preincubation also aggravated Aβ-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 Aβ. 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 Aβ-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aβ toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aβ 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 Aβ inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

Exercise-induced insulin-like growth factor I system concentrations after training in women.

INTRODUCTION This study examined the effects of short-term physical training on the acute hormonal response (i.e., growth hormone, total and free insulin-like growth factor I [IGF-I], and IGF binding proteins [IGFBP]-1, IGFBP-2, and IGFBP-3) to resistance exercise (RE) in women. METHODS Forty-six women (20.3 ± 0.3 yr, mass = 64.1 ± 7.3 kg, height = 165.7 ± 1.0 cm) were randomly assigned to an endurance training (E), resistance training (R), combined training (R + E), or control (C) group for 8wk. Subjects completed a standardized bout of RE (six sets of back squats at 10 repetition maximum) before and after training. Blood samples were obtained at rest (PRE), after the third set, immediately postexercise (POST), and at 15 min and 30 min after exercise. RESULTS Acute RE significantly increased (P < 0.05) serum growth hormone (mean ± SD; change from PRE to POST = +10.9 ± 7.5 μg·L-1), total IGF-I (+66.1 ± 25.4 μg·L-1), IGFBP-1 (+2.5 ± 3.1 μg·L-1), IGFBP-2 (+86.0 ± 86.8 μg·L-1), and IGFBP-3 (+0.69 ± 0.25 mg·L-1) concentrations and decreased free IGF-I concentrations (-0.14 ± 0.21 μg·L-1). After 8 wk of training, total IGF-I concentrations were significantly increased (change in POST concentrations from week 0 to week 8 = +82.5 ± 120.8 μg·L-1), and IGFBP-1 concentrations were significantly decreased (-6.7 ± 13.6 μg·L-1) during exercise in groups that participated in resistance training (R and R + E); no significant changes were seen after E or C. CONCLUSIONS Participation in resistance training increased total IGF-I and reduced IGFBP-1 concentrations during acute RE, indicating exercise mode-specific adaptations in the circulating IGF-I system.

Circulating bioactive and immunoreactive IGF-I remain stable in women, despite physical fitness improvements after 8 weeks of resistance, aerobic, and combined exercise training

Insulin-like growth factor-I (IGF-I) is regulated by a number of IGF-binding proteins (IGFBPs) and proteases that influence IGF-I bioactivity. A specific IGF-I kinase receptor activation assay (KIRA) has been developed that determines the ability of IGF-I to activate the IGF-I receptor by quantification of intracellular receptor autophosphorylation on IGF-I binding. KIRA-assessed IGF-I bioactivity has not been utilized within the context of chronic exercise training paradigms. This study measured total and free immunoreactive IGF-I, bioactive IGF-I, and IGFBP-1, -2, and -3 before (Pre), during (Mid), and after (Post) 8 wk of exercise training in young, healthy women, who were randomized into one of four groups: control (n = 10), resistance (n = 18), aerobic (n = 13), and combined (n = 15) exercise training. The training programs were effective in improving physical fitness specific to the exercise mode engaged in: increases were observed for lean mass ( approximately 2%), aerobic fitness (6-7%), and upper (20-24%) and lower (15-48%) body strength (all P values < 0.05). By contrast, no time, group, or interaction effects were observed for the circulating IGF-I system, as immunoreactive total (Pre = 264 +/- 16 microg/l; Mid = 268 +/- 17 microg/l; Post = 271 +/- 17 microg/l), free (Pre = 0.70 +/- 0.1 microg/l; Mid = 0.63 +/- 0.1 microg/l; Post = 0.63 +/- 0.2 microg/l) and bioactive (Pre = 2.35 +/- 0.3 microg/l; Mid = 2.25 +/- 0.3 microg/l; Post = 2.33 +/- 0.3 microg/l) IGF-I were unchanged throughout the study. All IGFBP measures were also unchanged. We conclude that increased lean mass, aerobic fitness, and upper and lower body strength resulting from an 8-wk exercise training programs can occur without concomitant increases in either circulating bioactive or immunoreactive IGF-I, as well as associated IGFBPs. In terms of reflecting positive anabolic neuromuscular outcomes, these data do not support a role for endocrine-derived IGF-I.

Effect of the data sampling rate on accuracy of indices for heart rate and blood pressure variability and baroreflex function in resting rats and mice

The aim of this study was to determine the minimal sampling rate (SR) required for blood pressure (BP) waveform recordings to accurately determine BP and heart rate (HR) variability indices and baroreceptor reflex sensitivity in rats and mice. We also determined if an 8-bit (versus 12-bit) analog-to-digital converter (ADC) resolution is sufficient to accurately determine these hemodynamic parameters and if spline interpolation to 1000 Hz of BP waveforms sampled at lower SRs can improve accuracy. BP and ECG recordings (1000 Hz SR, 12-bit ADC resolution) from two strains of rats and BP recordings (1000 Hz SR, 12-bit ADC resolution) from two strains of mice were mathematically converted to lower SRs and/or 8-bit ADC resolution. Time-domain HR variability and frequency-domain HR and BP variability indices and baroreflex sensitivity (using the sequence technique) were determined and the results obtained from the original files were compared to the results obtained from the mathematically altered files. Our results demonstrate that an ADC resolution of 8 bit is not sufficient to determine HR and BP variability in rats and mice and baroreceptor reflex sensitivity in mice. Average values for systolic, mean and diastolic BP and HR can be accurately derived from BP waveforms recorded at a minimal SR of 200 Hz in rats and mice. Spline interpolation of BP waveforms to 1000 Hz prior to extracting derived parameters reduces this minimal SR to 50 Hz in rats but still requires 200 Hz in mice. Frequency-domain BP variability (very low and low frequency spectral powers) can be estimated accurately at a minimum SR of 100 Hz in rats and mice and spline interpolation of BP waveforms to 1000 Hz reduces this minimal SR to 50 Hz in rats but does not reduce the minimal SR in mice. Time- and frequency-domain HR variability parameters require at least a SR of 1000 Hz in rats and mice. Spline interpolation of BP waveforms to 1000 Hz reduces this minimal SR to 100 Hz in rats and to 200 Hz in mice. Estimation of baroreflex sensitivity using the sequence technique requires a SR of at least 1000 Hz in rats and mice. Spline interpolation of BP waveforms to 1000 Hz reduces this minimal SR to 100 Hz in rats but does not reduce the minimum SR in mice. Finally, our results indicate that HR time series derived from BP waveforms are not totally consistent with HR time series derived from the ECG in rats. In conclusion, accurate assessment of HR variability and baroreflex sensitivity from BP waveform recordings requires a SR of at least 1000 Hz in rats and mice. If lower SRs are used for BP waveform recordings, a cubic spline interpolation to 1000 Hz (or an even higher SR) prior to extracting derived parameters significantly improves accuracy.

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.

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.

Effects of Acute and Chronic Exercise on Disulfide-Linked Growth Hormone Variants

PURPOSE To test the hypothesis that the appearance of disulfide-linked growth hormone (GH) aggregates during and after an acute resistance exercise test (ARET) in men could be influenced by chronic physical training. METHODS Fourteen men (28 +/- 1 yr) underwent two different 8-wk physical training programs designed to improve military performance. Before and after chronic training, subjects performed an ARET (six sets of 10 repetition-maximum squat) and had venous blood drawn pre-, mid-, and post-ARET (0, 15, and 30 min postexercise). To determine whether GH molecules were disulfide-linked, serum samples were chemically reduced via glutathione (GSH). Serum immunoreactive GH (IRGH) and immunofunctional GH (IFGH) concentrations were determined using two specific immunoassays, in nonreduced (-GSH) and reduced (+GSH) states. Data were analyzed using repeated-measures ANOVA. RESULTS No differences were observed in the GH responses of the two training programs; therefore, training group data were combined for analysis. GSH reduction increased the mean GH signal (-GSH: 1.4 +/- 0.3 microg x L(-1) vs +GSH: 1.7 +/- 0.3 microg x L(-1); P < 0.01) only when quantifying IRGH. Post hoc testing indicated that serum contained IRGH disulfide-linked GH aggregates at the mid, 0-, 15-, and 30-min posttime points of the ARET (P < 0.01), whereas GSH reduction did not affect IFGH concentrations. Chronic physical training had no effect on the ARET-induced GH response. CONCLUSION Acute resistance exercise leads to the appearance of disulfide-linked IRGH aggregates, and this response does not appear to be affected by 8 wk of chronic physical training. The physiological significance of increased proportions of disulfide-linked GH aggregates postexercise remains uncertain; however, structural alterations in GH moieties after acute exercise may represent important regulatory steps in mediating GH biological activity at selected target tissues.

Frequency response characteristics of whole body autoregulation of blood flow in rats

Previously, we demonstrated that very low-frequency (VLF) blood pressure variability (BPV) depends on voltage-gated L-type Ca(2+)-channels, suggesting that autoregulation of blood flow and/or myogenic vascular function significantly contributes to VLF BPV. To further substantiate this possibility, we tested the hypothesis that the frequency response characteristic of whole body autoregulation of blood flow is consistent with the frequency range of VLF BPV (0.02-0.2 Hz) in rats. In anesthetized rats (n = 11), BPV (0.016-0.5 Hz) was induced by computer-regulated cardiac pacing while blood pressure, heart rate, and cardiac output (CO) were recorded during control conditions (NaCl, 1 ml/h iv) and during alpha(1)-adrenergic receptor stimulation (phenylephrine, 1 mg.ml(-1).h(-1) iv) that has been reported to facilitate myogenic vascular function. Baroreceptor-heart rate reflex responses were elicited to confirm a functional baroreflex despite anesthesia. During control conditions, transfer function analyses between mean arterial pressure (MAP) and CO, and between MAP and total vascular conductance (CO/MAP) indicated autoregulation of blood flow at 0.016 Hz, passive vascular responses between 0.033 and 0.2 Hz, and vascular responses compatible with baroreflex-mediated mechanisms at 0.333 and 0.5 Hz. Stimulation of alpha(1)-adrenergic receptors extended the frequency range of autoregulation of blood flow to frequencies up to 0.033 Hz. In conclusion, depending on sympathetic vascular tone, whole body autoregulation of blood flow operates most effectively at frequencies below 0.05 Hz. This frequency range overlaps with the lower end of the frequency band of VLF BPV in rats. Baroreceptor reflex-like mechanisms contribute to LF (0.2-0.6 Hz) but not VLF BPV-induced vascular responses.

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.