Citations

The large conductance Ca2+-activated K+ (BK) channel β1-subunit (BK-β1) is a key modulator of BK channel electrophysiology and the downregulation of BK-β1 protein expression in vascular smooth muscle cells (SMCs) underlies diabetic vascular dysfunction. In this study, we hypothesized that the nuclear factor erythroid-2–related factor 2 (Nrf2) signaling pathway plays a significant role in the regulation of coronary BK channel function and vasodilation in high-fat diet (HFD)–induced obese/diabetic mice. We found that the protein expressions of BK-β1 and Nrf2 were markedly downregulated, whereas those of the nuclear factor-κB (NF-κB) and the muscle ring finger protein 1 (MuRF1 [a ubiquitin E3 ligase for BK-β1]) were significantly upregulated in HFD mouse arteries. Adenoviral expression of Nrf2 suppressed the protein expressions of NF-κB and MuRF1 but enhanced BK-β1 mRNA and protein expressions in cultured coronary SMCs. Knockdown of Nrf2 resulted in reciprocal changes of these proteins. Patch-clamp studies showed that coronary BK-β1–mediated channel activation was diminished in HFD mice. Importantly, the activation of Nrf2 by dimethyl fumarate significantly reduced the body weight and blood glucose levels of HFD mice, enhanced BK-β1 transcription, and attenuated MuRF1-dependent BK-β1 protein degradation, which in turn restored coronary BK channel function and BK channel–mediated coronary vasodilation in HFD mice. Hence, Nrf2 is a novel regulator of BK channel function with therapeutic implications in diabetic vasculopathy.

BK channels are major ionic determinants of vasodilation. BK channel function is impaired in diabetic vessels due to accelerated proteolysis of its beta-1 (BK-β1) subunits in response to increased oxidative stress. The nuclear factor E2-related factor-2 (Nrf2) signalling pathway has emerged as a master regulator of cellular redox status, and we hypothesized that it plays a central role in regulating BK channel function in diabetic vessels. We found that Nrf2 expression was markedly reduced in db/db diabetic mouse aortas, and this was associated with significant downregulation of BK-β1. In addition, the muscle ring finger protein 1 (MuRF1), a known E-3 ligase targeting BK-β1 ubiquitination and proteasomal degradation, was significantly augmented. These findings were reproduced by knockdown of Nrf2 by siRNA in cultured human coronary artery smooth muscle cells. In contrast, adenoviral transfer of Nrf2 gene in these cells downregulated MuRF1 and upregulated BK-β1 expression. Activation of Nrf2 by dimethyl fumarate preserved BK-β1 expression and protected BK channel and vascular function in db/db coronary arteries. These results indicate that expression of BK-β1 is closely regulated by Nrf2 and vascular BK channel function can be restored by Nrf2 activation. Nrf2 should be considered a novel therapeutic target in the treatment of diabetic vasculopathy.

Background: Hydrogen peroxide (H2O2)-induced oxidative stress has been demonstrated to induce afterdepolarizations and triggered activities in isolated myocytes, but the underlying mechanisms remain not fully understood. We aimed to explore whether protein kinase C (PKC) activation plays an important role in oxidative stress-induced afterdepolarizations. Methods: Action potentials and ion currents of isolated rabbit cardiomyocytes were recorded using the patch clamp technique. H2O2 (1 mM) was perfused to induce oxidative stress and the specific classical PKC inhibitor, Gö 6983 (1 μM), was applied to test the involvement of PKC. Results: H2O2 perfusion prolonged the action potential duration and induced afterdepolarizations. Pretreatment with Gö 6983 prevented the emergence of H2O2-induced afterdepolarizations. Additional application of Gö 6983 with H2O2 effectively suppressed H2O2-induced afterdepolarizations. H2O2 increased the late sodium current (INa,L) (n = 7, p < 0.01) and the L-type calcium current (ICa,L) (n = 5, p < 0.01), which were significantly reversed by Gö 6983 (p < 0.01). H2O2 also increased the transient outward potassium current (Ito) (n = 6, p < 0.05). However, Gö 6983 showed little effect on H2O2-induced enhancement of Ito. Conclusions: H2O2 induced afterdepolarizations via the activation of PKC and the enhancement of ICa,L and INa,L. These results provide evidence of a link between oxidative stress, PKC activation and afterdepolarizations.

Poly(GP) proteins are a promising pharmacodynamic marker for developing and testing therapeutics for treating C9ORF72 -associated amyotrophic lateral sclerosis.

On‐board magnetic resonance (MR) image guidance during radiation therapy offers the potential for more accurate treatment delivery. To utilize the real‐time image information, a crucial prerequisite is the ability to successfully segment and track regions of interest (ROI). The purpose of this work is to evaluate the performance of different segmentation algorithms using motion images (4 frames per second) acquired using a MR image‐guided radiotherapy (MR‐IGRT) system. Manual contours of the kidney, bladder, duodenum, and a liver tumor by an experienced radiation oncologist were used as the ground truth for performance evaluation. Besides the manual segmentation, images were automatically segmented using thresholding, fuzzy k‐means (FKM), k‐harmonic means (KHM), and reaction‐diffusion level set evolution (RD‐LSE) algorithms, as well as the tissue tracking algorithm provided by the ViewRay treatment planning and delivery system (VR‐TPDS). The performance of the five algorithms was evaluated quantitatively by comparing with the manual segmentation using the Dice coefficient and target registration error (TRE) measured as the distance between the centroid of the manual ROI and the centroid of the automatically segmented ROI. All methods were able to successfully segment the bladder and the kidney, but only FKM, KHM, and VR‐TPDS were able to segment the liver tumor and the duodenum. The performance of the thresholding, FKM, KHM, and RD‐LSE algorithms degraded as the local image contrast decreased, whereas the performance of the VP‐TPDS method was nearly independent of local image contrast due to the reference registration algorithm. For segmenting high‐contrast images (i.e., kidney), the thresholding method provided the best speed ( ) with a satisfying accuracy ( ). When the image contrast was low, the VR‐TPDS method had the best automatic contour. Results suggest an image quality determination procedure before segmentation and a combination of different methods for optimal segmentation with the on‐board MR‐IGRT system. PACS number(s): 87.57.nm, 87.57.N‐, 87.61.Tg

29 Background: Better biomarkers are needed in prostate cancer (PCa) to predict disease recurrence and guide optimal therapy. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor cancer phenotype impact PCa biology and clinical outcomes in localized disease. Methods: Genes associated with self-renewal, drug resistance, and tumorigenicity were analyzed by qRT-PCR on PCa mRNA from radical prostatectomy (RP) specimens (+/- disease recurrence). Wilcoxon rank sum two-sample test, multivariable recursive partitioning, and bootstrap internal validation measured and confirmed associations with recurrence. Further validation was conducted in external cohorts and in-silico, as well as in vitro and in vivo using siRNA knockdown and lentiviral overexpression to determined the effect of gene expression on PCa proliferation, invasion and tumor growth. Results: Four candidate genes were differentially expressed in PCa recurrence, and of these, low AXIN2 expression was internally validated. Validation in external cohorts demonstrated low AXIN2 expression was associated with more aggressive prostate cancer and was independently associated with biochemical recurrence (BCR) and metastasis-free survival (MFS) after RP. In vitro, low AXIN2 expression was associated with a cancer stem-like cell-surface signature, and siRNA knockdown of AXIN2 resulted in significantly greater invasiveness. Conversely, ectopic overexpression of AXIN2 significantly reduced cell proliferation and tumor growth in mice. Conclusions: Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts. AXIN2 expression levels in PCa cells significantly impacted invasiveness, proliferation and tumor growth. AXIN2 represents a putative biomarker and potential therapeutic target in early prostate cancer.