Repurposing hyperpolarization-activated cyclic nucleotide-gated channels as a novel therapy for breast cancer.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated cation channel family known to be expressed in the heart and central nervous system. Ivabradine, a small molecule HCN channel-blocker, is FDA-approved for clinical use as a heart rate-reducing agent. We found that HCN2 and HCN3 are overexpressed in breast cancer cells compared with normal breast epithelia, and the high expression of HCN2 and HCN3 is associated with poorer survival in breast cancer patients. Inhibition of HCN by Ivabradine or by RNAi, aborted breast cancer cell proliferation in vitro and suppressed tumour growth in patient-derived tumour xenograft models established from triple-negative breast cancer (TNBC) tissues, with no evident side-effects on the mice. Transcriptome-wide analysis showed enrichment for cholesterol metabolism and biosynthesis as well as lipid metabolism pathways associated with ER-stress following Ivabradine treatment. Mechanistic studies confirmed that HCN inhibition leads to ER-stress, in part due to disturbed Ca2+ homeostasis, which subsequently triggered the apoptosis cascade. More importantly, we investigated the synergistic effect of Ivabradine and paclitaxel on TNBC and confirmed that both drugs acted synergistically in vitro through ER-stress to amplify signals for caspase activation. Combination therapy could suppress tumour growth of xenografts at much lower doses for both drugs. In summary, our study identified a new molecular target with potential for being developed into targeted therapy, providing scientific grounds for initiating clinical trials for a new treatment regimen of combining HCN inhibition with chemotherapy.

Hydroxycarbamide treatment in children with Sickle Cell Anaemia is associated with more intact white matter integrity: a quantitative MRI study.

Sickle cell anaemia (SCA) is a devastating genetic blood disorder leading to chronic anaemia, impaired cerebrovascular dilatory capacity and cerebral infarctions. Our aim was to assess the relationship between microstructural properties of the white matter (WM) and both cerebrovascular reactivity (CVR) and cerebral blood flow, as well as the effects of hydroxycarbamide on these relationships. Our results demonstrate that mean CVR was increased in hydroxycarbamide-treated patients compared to untreated patients. Moreover, untreated SCA patients had increased skew and kurtosis of mean diffusivity histograms in the WM compared to hydroxycarbamide-treated patients and healthy age-matched controls, indicating disruption of WM integrity. Regression analysis of CVR and WM mean diffusivity (MD) revealed a significant linear relationship between CVR and MD histogram skew and kurtosis in healthy controls, but not in either of the two SCA groups. These findings suggest that patients treated with hydroxycarbamide possess white matter MD histogram parameters which more closely resemble those of healthy controls.

Quantification of pathophysiological alterations in venous oxygen saturation: A comparison of global MR susceptometry techniques.

The purpose of this study was to compare the Infinite Cylinder and Forward Field methods of quantifying global venous oxygen saturation (Yv) in the superior sagittal sinus (SSS) from MRI phase data, and assess their applicability in systemic cerebrovascular disease.15 children with sickle cell disease (SCD) and 10 healthy age-matched controls were imaged on a 3.0 T MRI system. Anatomical and phase data around the superior sagittal sinus were acquired from a clinically available susceptibility weighted imaging sequence and converted to Yv using the Infinite Cylinder and Forward Field methods. Yv was significantly higher when calculated using the Infinite Cylinder method compared to the Forward Field method in both patients (p = 0.003) and controls (p < 0.001). A significant difference in Yv was observed between patients and controls for the Forward Field method only (p = 0.006). While various implementations of Yv quantification can be used in practice, the results can differ significantly. Simplistic models such as the Infinite Cylinder method may be easier to implement, but their dependence on broad assumptions can lead to an overestimation of Yv, and may reduce the sensitivity to pathophysiological changes in Yv.

Assessment of cerebral blood flow with magnetic resonance imaging in children with sickle cell disease: A quantitative comparison with transcranial Doppler ultrasonography.

Introduction: Transcranial Doppler ultrasonography (TCD) is a clinical tool for stratifying ischemic stroke risk by identifying abnormal elevations in blood flow velocity (BFV) in the middle cerebral artery (MCA). However, TCD is not effective at screening for subtle neurologic injury such as silent cerebral infarcts. To better understand this disparity, we compared TCD measures of BFV with tissue-level cerebral blood flow (CBF) using arterial spin-labeling MRI in children with and without sickle cell disease, and correlated these measurements against clinical hematologic measures of disease severity. Methods: TCD and MRI assessment were performed in 13 pediatric sickle cell disease patients and eight age-matched controls. Using MRI measures of MCA diameter and territory weight, TCD measures of BFV in the MCA [cm/s] were converted into units of CBF [ml min-1100 g-1] for comparison. Results: There was no significant association between TCD measures of BFV in the MCA and corresponding MRI measures of CBF in patients (r = .28, p = .39) or controls (r = .10, p = .81). After conversion from BFV into units of CBF, a strong association was observed between TCD and MRI measures (r = .67, p = .017 in patients, r = .86, p = .006 in controls). While BFV in the MCA showed a lack of correlation with arterial oxygen content, an inverse association was observed for CBF measurements. Conclusions: This study demonstrates that BFV in the MCA cannot be used as a surrogate marker for tissue-level CBF in children with sickle cell disease. Therefore, TCD alone may not be sufficient for understanding and predicting subtle pathophysiology in this population, highlighting the potential clinical value of tissue-level CBF.

The Potential for Advanced Magnetic Resonance Neuroimaging Techniques in Pediatric Stroke Research.

BACKGROUND: This article was written to provide clinicians and researchers with an overview of a number of advanced neuroimaging techniques in an effort to promote increased utility and the design of future studies using advanced neuroimaging in childhood stroke. The current capabilities of advanced magnetic resonance imaging techniques provide the opportunity to build on our knowledge of the consequences of stroke on the developing brain. These capabilities include providing information about the physiology, metabolism, structure, and function of the brain that are not routinely evaluated in the clinical setting. METHODS: During the Proceedings of the Stroke Imaging Laboratory for Children Workshop in Toronto in June 2015, a subgroup of clinicians and imaging researchers discussed how the application of advanced neuroimaging techniques could further our understanding of the mechanisms of stroke injury and repair in the pediatric population. This subgroup was established based on their interest and commitment to design collaborative, advanced neuroimaging studies in the pediatric stroke population. RESULTS: In working toward this goal, we first sought to describe here the magnetic resonance imaging techniques that are currently available for use, and how they have been applied in other stroke populations (e.g., adult and perinatal stroke). CONCLUSIONS: With the continued improvement in advanced neuroimaging techniques, including shorter acquisition times, there is an opportunity to apply these techniques to their full potential in the research setting and learn more about the effects of stroke in the developing brain.

The severity of anaemia depletes cerebrovascular dilatory reserve in children with sickle cell disease: a quantitative magnetic resonance imaging study.

Overt ischaemic stroke is one of the most devastating complications in children with sickle cell disease (SCD). The compensatory response to anaemia in SCD includes an increase in cerebral blood flow (CBF) by accessing cerebrovascular dilatory reserve. Exhaustion of dilatory reserve secondary to anaemic stress may lead to cerebral ischaemia. The purpose of this study was to investigate CBF and cerebrovascular reactivity (CVR) using magnetic resonance imaging (MRI) in children with SCD and to correlate these with haematological markers of anaemia. Baseline CBF was measured using arterial spin labelling. Blood-oxygen level-dependent MRI in response to a CO2 stimulus was used to acquire CVR. In total, 28 children with SCD (23 not on any disease-modifying treatment, 5 on chronic transfusion) and 22 healthy controls were imaged using MRI. Transfusion patients were imaged at two time points to assess the effect of changes in haematocrit after a transfusion cycle. In children with SCD, CBF was significantly elevated compared to healthy controls, while CVR was significantly reduced. Both measures were significantly correlated with haematocrit. For transfusion patients, CBF decreased and CVR increased following a transfusion cycle. Lastly, a significant correlation was observed between CBF and CVR in both children with SCD and healthy controls.

MRI-based cerebrovascular reactivity using transfer function analysis reveals temporal group differences between patients with sickle cell disease and healthy controls.

OBJECTIVES: Cerebrovascular reactivity (CVR) measures the ability of cerebral blood vessels to change their diameter and, hence, their capacity to regulate regional blood flow in the brain. High resolution quantitative maps of CVR can be produced using blood-oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) in combination with a carbon dioxide stimulus, and these maps have become a useful tool in the clinical evaluation of cerebrovascular disorders. However, conventional CVR analysis does not fully characterize the BOLD response to a stimulus as certain regions of the brain are slower to react to the stimulus than others, especially in disease. Transfer function analysis (TFA) is an alternative technique that can account for dynamic temporal relations between signals and has recently been adapted for CVR computation. We investigated the application of TFA in data on children with sickle cell disease (SCD) and healthy controls, and compared them to results derived from conventional CVR analysis. MATERIALS AND METHODS: Data from 62 pediatric patients with SCD and 34 age-matched healthy controls were processed using conventional CVR analysis and TFA. BOLD data were acquired on a 3 Tesla MRI scanner while a carbon dioxide stimulus was quantified by sampling the end-tidal partial pressures of each exhaled breath. In addition, T1 weighted structural imaging was performed to identify grey and white matter regions for analysis. The TFA method generated maps representing both the relative magnitude change of the BOLD signal in response to the stimulus (Gain), as well as the BOLD signal speed of response (Phase) for each subject. These were compared to CVR maps calculated from conventional analysis. The effect of applying TFA on data from SCD patients versus controls was also examined. RESULTS: The Gain measures derived from TFA were significantly higher than CVR values based on conventional analysis in both SCD patients and healthy controls, but the difference was greater in the SCD data. Moreover, while these differences were uniform across the grey and white matter regions of controls, they were greater in white matter than grey matter in the SCD group. Phase was also shown to be significantly correlated with the amount that TFA increases CVR estimates in both the grey and white matter. CONCLUSIONS: We demonstrated that conventional CVR analysis underestimates vessel reactivity and this effect is more prominent in patients with SCD. By using TFA, the resulting Gain and Phase measures more accurately characterize the BOLD response as it accounts for the temporal dynamics responsible for the CVR underestimation. We suggest that the additional information offered through TFA can provide insight into the mechanisms underlying CVR compromise in cerebrovascular diseases.

Reduced cerebrovascular reserve is regionally associated with cortical thickness reductions in children with sickle cell disease.

Sickle cell disease (SCD) is a genetic disorder which adversely affects cerebrovascular health. Previous studies have demonstrated regional cortical thinning in SCD. However, the reason behind regional reductions in cortical thickness remains unclear. Therefore, we aimed to explore the possible link between the state of cerebrovascular health and cortical thickness. In this study, we obtained magnetic resonance (MR) based measures of cerebrovascular reactivity (CVR), a measure of vascular health, and cortical thickness in SCD patients (N=60) and controls of similar age and similar gender ratio (N=27). The group comparison analysis revealed significant regionally specific reductions in CVR and cortical thickness in the SCD group compared to the controls. In addition, a regional association analysis was performed between CVR and cortical thickness in the SCD group which revealed a significant regional association in several brain regions with the highest strength of association observed in the left cuneus, right post central gyrus and the right temporal pole. The regional association analysis revealed that significant associations were found in brain regions with high metabolic activity (anterior cingulate, posterior cingulate, occipital gyrus, precuneus) thus demonstrating that these regions could be most vulnerable to structural damage under hypoxic conditions.

Developmental trajectories of cerebrovascular reactivity in healthy children and young adults assessed with magnetic resonance imaging.

KEY POINTS: Cerebrovascular reactivity (CVR) reflects the vasodilatory reserve of cerebral resistance vessels. Normal development in children is associated with significant changes in blood pressure, cerebral blood flow (CBF) and cerebral oxygen metabolism. Therefore, it stands to reason that CVR will also undergo changes during this period. The study acquired magnetic resonance imaging measures of CVR and CBF in healthy children and young adults to trace their changes with age. We found that CVR changes in two phases, increasing with age until the mid-teens, followed by a decrease. Baseline CBF declined steadily with age. We conclude that CVR varies with age during childhood, which prompts future CVR studies involving children to take into account the effect of development. ABSTRACT: Cerebrovascular reactivity (CVR) reflects the vasculature's ability to accommodate changes in blood flow demand thereby serving as a critical imaging tool for mapping vascular reserve. Normal development is associated with extensive physiological changes in blood pressure, cerebral blood flow and cerebral metabolic rate of oxygen, all of which can affect CVR. Moreover, the evolution of these physiological parameters is most prominent during childhood. Therefore, the aim of this study was to use non-invasive magnetic resonance imaging (MRI) to characterize the developmental trajectories of CVR in healthy children and young adults, and relate them to changes in cerebral blood flow (CBF). Thirty-four healthy subjects (17 males, 17 females; age 9-30 years) underwent CVR assessment using blood oxygen level-dependent MRI in combination with a computer controlled CO2 stimulus. In addition, baseline CBF was measured with a pulsed arterial spin labelling sequence. CVR exhibited a gradual increase with age in both grey and white matter up to 14.7 years. After this break point, a negative correlation with age was detected. Baseline CBF maintained a consistent negative linear correlation across the entire age range. The significant age-dependent changes in CVR and CBF demonstrate the evolution of cerebral haemodynamics in children and should be taken into consideration. The shift in developmental trajectory of CVR from increasing to decreasing suggests that physiological factors beyond baseline CBF also influence CVR.

Reproducibility of cerebrovascular reactivity measures in children using BOLD MRI.

PURPOSE: To evaluate the reproducibility of cerebrovascular reactivity (CVR) measurements acquired in children using magnetic resonance imaging (MRI) in combination with a computer-controlled carbon dioxide (CO2 ) stimulus. MATERIALS AND METHODS: Ten healthy children (age 16.1 ± 1.6 years) underwent CVR imaging on a 3T scanner using a blood-oxygen level-dependent (BOLD) MRI sequence. Targeted hypercapnia was induced during imaging with a CO2 gas challenge delivered using a specialized gas sequencer (RespirAct). A total of four BOLD scans were performed over 2 separate days to test within-day and between-day consistency of the data. CVR values were computed by correlating the relative change in BOLD signal in response to the CO2 stimulus delivered to the each subject. RESULTS: Intraclass correlation coefficients (ICCs) of within-day values show highly reproducible measures in both the gray matter (ICC = 0.857, P < 0.001) and white matter (ICC = 0.895, P < 0.001). Relatively lower between-day reproducibility was observed in both the gray matter (ICC = 0.776, P = 0.001) and white matter (ICC = 0.719, P = 0.004). CONCLUSION: Using a computer-controlled CO2 stimulus, we have demonstrated the reliability of BOLD-CVR measurements in pediatric subjects. Within-day and between-day metrics of reproducibility were comparable to adult data.

Dynamic contrast-enhanced MRI and CT provide comparable measurement of blood-brain barrier permeability in a rodent stroke model.

In the current management of acute ischemic stroke (AIS), clinical criteria are used to estimate the risk of hemorrhagic transformation (HT), which is a devastating early complication. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and computed tomography (DCE-CT) may serve as physiologically-based decision making tools to more reliably assess the risk of HT. Before these tools can be properly validated, the comparability of the blood-brain barrier (BBB) permeability measurements they generate should be assessed. Sixteen rats were subjected to a transient middle cerebral artery occlusion before successively undergoing DCE-CT and DCE-MRI at 24-hours. BBB permeability (K(trans)) values were generated from both modalities. A correlation of R=0.677 was found (p<0.01) and the resulting relationship was [DCE-CT=(0.610*DCE-MRI)+4.140]. A variance components analysis found the intra-rat coefficient of variation to be 0.384 and 0.258 for K(trans) values from DCE-MRI and DCE-CT respectively. Permeability measures from DCE-CT were 22% higher than those from DCE-MRI. The results of this study demonstrate for the first time comparability between DCE-CT and DCE-MRI in the assessment of AIS. These results may provide a foundation for future clinical trials making combined use of these modalities.

Longitudinal assessment of imatinib's effect on the blood-brain barrier after ischemia/reperfusion injury with permeability MRI.

Acute ischemic stroke (AIS) often results in degeneration of the blood-brain barrier (BBB), which can lead to vasogenic edema and an increased risk of intracerebral hemorrhage. Imatinib is an agent that may be able to protect the BBB and reduce the risk of the harmful consequences of BBB degeneration. We sought to measure the effect of Imatinib on the BBB after experimental stroke longitudinally in vivo with permeability dynamic contrast-enhanced MRI. Ischemia/reperfusion injury was induced with a transient middle cerebral artery occlusion surgery. Rats were given Imatinib at 2 and 20 h after stroke onset. Post-assessment included neurologic functioning, MR imaging, Evans Blue extravasation, Western blot, and immunohistology assay. Imatinib protected the BBB by 24 h but failed to decrease BBB permeability at an earlier time-point. Imatinib also reduced infarct volume, edema, and improved neurologic functioning by 24 h. Rats treated with Imatinib also had a higher expression of the BBB structural protein Zona ocludens-1 and a reduction in nuclear factor-kappa beta (NF-κß) activation. Imatinib is a promising agent to protect the BBB after AIS, but its effect on the BBB may not become prominent until 24 h after the onset of ischemia. This finding may help elucidate Imatinib's role in the clinical management of AIS and influence future study designs.

Assessment of intracranial blood flow velocities using a computer controlled vasoactive stimulus: a comparison between phase contrast magnetic resonance angiography and transcranial Doppler ultrasonography.

PURPOSE: To compare measurements of blood flow velocity (BFV) and BFV changes in the middle cerebral arteries (MCA) acquired from phase contrast magnetic resonance angiography (PCMRA) and transcranial Doppler ultrasound (TCD) during controlled manipulation of end-tidal partial pressure of carbon dioxide (PetCO2 ). MATERIALS AND METHODS: In vivo TCD and PCMRA velocity data from the M1 segment in the MCA of nine healthy adult volunteers were acquired during precise targeting of PetCO2 induced by a computer-controlled gas delivery system. Doppler spectra and phase contrast data were processed into time-averaged peak-velocity (TAPV) values for comparison. Changes in velocity between baseline and hypercapnia were analyzed in terms of velocity-based cerebrovascular reactivity (CVR). RESULTS: Good correlation between the pairs of velocity measurements acquired from the two modalities were found (ρ = 0.81), but Bland-Altman analysis indicates a significant bias error. There was relatively weak agreement between the pairs of computed CVR values (ρ = -0.26). CONCLUSION: Under precise PetCO2 control, PCMRA proves to be more consistent than TCD. Despite issues with variability, TCD is qualitatively comparable to PCMRA measures of velocity in the MCA. However, PCMRA velocity results are better suited for analyses that require quantitative values, such as CVR.

Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid inhibits hepatocellular carcinoma in vitro and in vivo via stabilizing IkBα.

Ent-11-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) isolated from Pteris Semipinnata L is known to inhibit certain tumor cells in vitro. The information on the in vivo effect of 5F is limited and its effect on hepatocellular carcinoma (HCC) is unknown. In this study, the anti-tumor effect of 5F was investigated in a diethylnitrosamine (DEN)-induced mouse HCC model. In addition to therapeutic effect, the potential side effect was monitored. A panel of cultured HCC cells was used to confirm the in vivo data and explore the responsible molecular pathway. The result showed that 5F significantly inhibited the DEN-induced HCC tumors by reducing the number of tumor foci and the volume of tumors. Furthermore, 5F induced the death of cultured HCC cells in dose- and time-dependent manners. The cell death was confirmed to be apoptotic by in vivo and in vitro TUNEL assays. 5F inhibited NF-kB by stabilizing its inhibitor IkBα, reducing the nuclear p65 and inhibiting NF-kB activity. Subsequently it affected the NF-kB downstream molecules with a decrease in anti-apoptotic Bcl-2 and increase in pro-apoptotic Bax and Bak. During the whole period of the experiment, mice receiving 5F appeared to be healthy, though they suffered from a mild degree of hair loss. 5F did not damage liver and renal functions. In conclusion, 5F is effective against HCC with minimal side effects. It induces apoptosis in HCC cells via inhibiting NF-kB, leading to the decrease of Bcl-2 but the increase of Bax and Bak.

Anticancer efficacy of 5F in NNK-induced lung cancer development of A/J mice and human lung cancer cells.

The mechanism responsible for the apoptotic effect induced by ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) is not fully understood and its in vivo effect has not been tested. In this study, the effect and mechanism of 5F was investigated in cigarette smoking carcinogen 4-methylnitrosamino-1-3-pyridyl-butanone (NNK)-induced mouse lung tumor model and in cultured lung cancer cells NCI-H23 and CRL-2066. 5F were given to mice after they were treated with NNK for 18 weeks. The effect of 5F on the lung tumor formation was examined, and its side effect was monitored. Cell proliferation and apoptosis were determined through expression of PCNA, Bcl-2, Bax, and TUNEL assay in in vivo animal model. 5F significantly inhibited the NNK-induced lung tumors by inducing apoptosis and suppressing cell proliferation in vivo with minimal side effects. Cell culture experiments showed that 5F translocated Bax into the mitochondria, downregulated Bcl-2, activated caspase-9 and caspase-3, released cytochrome c into the cytosol, and translocated AIF from the mitochondria to the nucleus, which leading to G2-M cell cycle arrest and cell apoptosis. 5F also activated ERK1/2 and the inhibition of ERK1/2 suppressed 5F-mediated changes in apoptotic molecules. In addition to ERK1/2, 5F activated Akt. The inhibition of Akt further facilitated the apoptosis induced, suggesting that Akt activation was anti-apoptotic rather than pro-apoptotic. Collectively, 5F is effective against lung cancer in vivo with minimal side effects. It induces apoptosis in lung cancer through the mitochondrial-mediated pathway, in which the activation of ERK is critical.