Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma.

Excessive ROS promote allergic asthma, a condition characterized by airway inflammation, eosinophilic inflammation, and increased airway hyperreactivity (AHR). The mechanisms by which airway ROS are increased and the relationship between increased airway ROS and disease phenotypes are incompletely defined. Mitochondria are an important source of cellular ROS production, and our group discovered that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is present in mitochondria and activated by oxidation. Furthermore, mitochondrial-targeted antioxidant therapy reduced the severity of allergic asthma in a mouse model. Based on these findings, we developed a mouse model of CaMKII inhibition targeted to mitochondria in airway epithelium. We challenged these mice with OVA or Aspergillus fumigatus. Mitochondrial CaMKII inhibition abrogated AHR, inflammation, and eosinophilia following OVA and A. fumigatus challenge. Mitochondrial ROS were decreased after agonist stimulation in the presence of mitochondrial CaMKII inhibition. This correlated with blunted induction of NF-κB, the NLRP3 inflammasome, and eosinophilia in transgenic mice. These findings demonstrate a pivotal role for mitochondrial CaMKII in airway epithelium in mitochondrial ROS generation, eosinophilic inflammation, and AHR, providing insights into how mitochondrial ROS mediate features of allergic asthma.

Acid-Sensing Ion Channel 1a Contributes to Airway Hyperreactivity in Mice.

Neurons innervating the airways contribute to airway hyperreactivity (AHR), a hallmark feature of asthma. Several observations suggested that acid-sensing ion channels (ASICs), neuronal cation channels activated by protons, might contribute to AHR. For example, ASICs are found in vagal sensory neurons that innervate airways, and asthmatic airways can become acidic. Moreover, airway acidification activates ASIC currents and depolarizes neurons innervating airways. We found ASIC1a protein in vagal ganglia neurons, but not airway epithelium or smooth muscle. We induced AHR by sensitizing mice to ovalbumin and found that ASIC1a-/- mice failed to exhibit AHR despite a robust inflammatory response. Loss of ASIC1a also decreased bronchoalveolar lavage fluid levels of substance P, a sensory neuropeptide secreted from vagal sensory neurons that contributes to AHR. These findings suggest that ASIC1a is an important mediator of AHR and raise the possibility that inhibiting ASIC channels might be beneficial in asthma.

CaMKII inhibition in type II pneumocytes protects from bleomycin-induced pulmonary fibrosis by preventing Ca2+-dependent apoptosis.

The calcium and calmodulin-dependent kinase II (CaMKII) translates increases in intracellular Ca(2+) into downstream signaling events. Its function in pulmonary pathologies remains largely unknown. CaMKII is a well-known mediator of apoptosis and regulator of endoplasmic reticulum (ER) Ca(2+). ER stress and apoptosis of type II pneumocytes lead to aberrant tissue repair and progressive collagen deposition in pulmonary fibrosis. Thus we hypothesized that CaMKII inhibition alleviates fibrosis in response to bleomycin by attenuating apoptosis and ER stress of type II pneumocytes. We first established that CaMKII was strongly expressed in the distal respiratory epithelium, in particular in surfactant protein-C-positive type II pneumocytes, and activated after bleomycin instillation. We generated a novel transgenic model of inducible expression of the CaMKII inhibitor peptide AC3-I limited to type II pneumocytes (Tg SPC-AC3-I). Tg SPC-AC3-I mice were protected from development of pulmonary fibrosis after bleomycin exposure compared with wild-type mice. CaMKII inhibition also provided protection from apoptosis in type II pneumocytes in vitro and in vivo. Moreover, intracellular Ca(2+) levels and ER stress were increased by bleomycin and significantly blunted with CaMKII inhibition in vitro. These data demonstrate that CaMKII inhibition prevents type II pneumocyte apoptosis and development of pulmonary fibrosis in response to bleomycin. CaMKII inhibition may therefore be a promising approach to prevent or ameliorate the progression of pulmonary fibrosis.

Mitochondrial-targeted antioxidant therapy decreases transforming growth factor-ß-mediated collagen production in a murine asthma model.

Asthma is a disease of acute and chronic inflammation in which cytokines play a critical role in orchestrating the allergic inflammatory response. IL-13 and transforming growth factor (TGF)-ß promote fibrotic airway remodeling, a major contributor to disease severity. Improved understanding is needed, because current therapies are inadequate for suppressing development of airway fibrosis. IL-13 is known to stimulate respiratory epithelial cells to produce TGF-ß, but the mechanism through which this occurs is unknown. Here, we tested the hypothesis that reactive oxygen species (ROS) are a critical signaling intermediary between IL-13 or allergen stimulation and TGF-ß-dependent airway remodeling. We used cultured human bronchial epithelial cells and an in vivo mouse model of allergic asthma to map a pathway where allergens enhanced mitochondrial ROS, which is an essential upstream signal for TGF-ß activation and enhanced collagen production and deposition in airway fibroblasts. We show that mitochondria in airway epithelium are an essential source of ROS that activate TGF-ß expression and activity. TGF-ß from airway epithelium stimulates collagen expression in fibroblasts, contributing to an early fibrotic response to allergen exposure in cultured human airway cells and in ovalbumin-challenged mice. Treatment with the mitochondrial-targeted antioxidant, (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), significantly attenuated mitochondrial ROS, TGF-ß, and collagen deposition in OVA-challenged mice and in cultured human epithelial cells. Our findings suggest that mitochondria are a critical source of ROS for promoting TGF-ß activity that contributes to airway remodeling in allergic asthma. Mitochondrial-targeted antioxidants may be a novel approach for future asthma therapies.

Oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulates vascular smooth muscle migration and apoptosis.

Activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and reactive oxygen species (ROS) promote neointimal hyperplasia after vascular injury. CaMKII can be directly activated by ROS through oxidation. In this study, we determined whether abolishing the oxidative activation site of CaMKII alters vascular smooth muscle cell (VCMC) proliferation, migration and apoptosis in vitro and neointimal formation in vivo. VSMC isolated from a knock-in mouse with oxidation-resistant CaMKIIδ (CaMKII M2V) displayed similar proliferation but decreased migration and apoptosis. Surprisingly, ROS production and expression of the NADPH oxidase subunits p47 and p22 were decreased in M2V VSMC, whereas superoxide dismutase 2 protein expression was upregulated. In vivo, after carotid artery ligation, no differences in neointimal size or remodeling were observed. In contrast to VSMC, CaMKII expression and autonomous activity were significantly higher in M2V compared to WT carotid arteries, suggesting that an autoregulatory mechanism determines CaMKII activity in vivo. Our findings demonstrate that preventing oxidative activation of CaMKII decreases migration and apoptosis in vitro and suggest that CaMKII regulates ROS production. Our study presents novel evidence that CaMKII expression in vivo is regulated by a negative feedback loop following oxidative activation.

CaMKII is essential for the proasthmatic effects of oxidation.

Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.

Pain after percutaneous radiofrequency ablation of renal tumors.

BACKGROUND AND PURPOSE: Most patients have minimal pain after percutaneous radiofrequency ablation (RFA) of a renal tumor. However, anecdotally, there is some variation in the amount of patient discomfort. Our goal was to identify relevant patient factors and characteristics of their renal tumors that may influence pain after percutaneous RF ablation. PATIENTS AND METHODS: We performed a retrospective chart review of 59 sequential patients who received percutaneous RFA between 2001 and 2005 at a single institution. Data on patient age, sex, body mass index (BMI), and narcotic administration in the periprocedural period were available for 46 patients. Preoperative imaging (CT or MRI) was reviewed to determine tumor size and location, as well as the shortest distance of the mass to the body-wall musculature. RESULTS: The distance from the renal mass to the body-wall musculature was significantly correlated with the total narcotics received in the periprocedural period. This measured distance did not correlate with the patient's BMI. No other relations between patient factors or tumor characteristics and peri-procedural narcotic usage were identified. CONCLUSION: Patients whose tumors lie close to their body-wall musculature have greater narcotic requirements in the periprocedural period. Knowledge of this correlation should result in better patient counseling and help anticipate periprocedural analgesia requirements.

Contemporary laparoscopic and open radical retropubic prostatectomy: pathologic outcomes and Kattan postoperative nomograms are equivalent.

OBJECTIVES: Although contemporary cohort comparisons are lacking, open surgeons have questioned the oncologic efficacy of laparoscopic radical prostatectomy (LRP) owing to the lack of haptic feedback. As such, we compared the pathologic outcomes and predicted the progression-free survival after open radical prostatectomy (ORP) and LRP in a single referral setting within a defined period, thereby isolating the effect of the surgical technique alone. METHODS: Data were collected on 169 ORPs and 111 LRPs performed at our institution from May 2003 to May 2005. The surgical pathologic outcomes were compared, and the Kattan postoperative nomogram was used to calculate the 5 and 7-year progression-free probabilities after ORP and LRP. RESULTS: On univariate analysis, no differences were found in age, Gleason sum, stage, margin status, extracapsular extension, and seminal vesicle invasion. The positive margin rate was 29% for LRP and 35% for ORP (P = 0.29), and no difference was found even after stratifying by pathologic stage. After controlling for Gleason sum, extracapsular extension, seminal vesicle invasion, nodal involvement, margin status, and preoperative prostate-specific antigen, no difference was found in detectable prostate-specific antigen after ORP and LRP (P = 0.73). When the Kattan postoperative nomogram was used to compute the biochemical progression-free probabilities, no differences were found at 5 (P = 0.51) and 7 years (P = 0.50). CONCLUSIONS: In this contemporary series without the confounding effects of stage migration, regional practice variation, or the use of historical controls, the pathologic outcome after conventional LRP was similar to that after ORP. Biochemical progression-free survival was also similar using a validated multivariate predictive model.

Optimal timing of acustimulation for antiemetic prophylaxis as an adjunct to ondansetron in patients undergoing plastic surgery.

We designed this study to evaluate the antiemetic efficacy of transcutaneous electrical acupoint stimulation in combination with ondansetron when applied before, after, or both before and after plastic surgery. A randomized, double-blind, sham-controlled study design was used to compare three prophylactic acustimulation treatment schedules: preoperative--an active device was applied for 30 min before and a sham device for 72 h after surgery; postoperative--a sham device was applied for 30 min before and an active device for 72 h after surgery; and perioperative--an active device was applied for 30 min before and 72 h after surgery (n = 35 per group). All patients received a standardized general anesthetic, and ondansetron 4 mg IV was administered at the end of surgery. The incidence of vomiting/retching and the need for rescue antiemetics were determined at specific time intervals for up to 72 h after surgery. Nausea scores were recorded with an 11-point verbal rating scale. Other outcome variables assessed included discharge times (for outpatients), resumption of normal activities of daily living, complete antiemetic response rate, and patient satisfaction with antiemetic therapy and quality of recovery. Perioperative use of the ReliefBand significantly increased complete responses (68%) compared with use of the device before surgery only (43%). Median postoperative nausea scores were significantly reduced in the peri- and postoperative (versus preoperative) treatment groups. Finally, patient satisfaction with the quality of recovery (83 +/- 16 and 85 +/- 13 vs 72 +/- 18) and antiemetic management (96 +/- 9 and 94 +/- 10 vs 86 +/- 13) on an arbitrary scale from 0 = worst to 100 = best was significantly higher in the groups receiving peri- or postoperative (versus preoperative) acustimulation therapy. For patients discharged on the day of surgery, the time to home readiness was significantly reduced (114 +/- 41 min versus 164 +/- 50 min; P < 0.05) when acustimulation was administered perioperatively (versus preoperatively). In conclusion, acustimulation with the ReliefBand was most effective in reducing postoperative nausea and vomiting and improving patients' satisfaction with their antiemetic therapy when it was administered after surgery.