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1.  CaMKII Is Essential for the Proasthmatic Effects of Oxidation 
Science translational medicine  2013;5(195):195ra97.
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 Ca2+/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 hyper-reactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent pro-asthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
PMCID: PMC4331168  PMID: 23884469
2.  Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides 
Cardiovascular Research  2013;101(1):165-174.
Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca2+ handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined.
Methods and results
We examined the effects of digitoxin (DGT) on Ca2+ handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca2+ waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated.
These results suggest that the arrhythmogenic adverse effects of CGs on Ca2+ handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
PMCID: PMC3868350  PMID: 24104877
Calcium; Reactive oxygen species; NADPH oxidase; Mitochondria; CaMKII
3.  Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance 
Modulation of central tolerance through RANKL alters thymic output and enhances anti-tumor immunity.
Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.
PMCID: PMC4010907  PMID: 24752296
4.  Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice 
Nature neuroscience  2014;17(5):694-703.
Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, which display mutant huntingtin in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models is not associated with classical astrogliosis, but is associated with decreased Kir4.1 K+ channel functional expression, leading to elevated in vivo levels of striatal extracellular K+, which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K+, recovered aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte–mediated K+ homeostasis, revealing astrocytes and Kir4.1 channels as novel therapeutic targets.
PMCID: PMC4064471  PMID: 24686787
5.  Secretion of atypical protein subtrates by the ESAT-6 Secretion System of Staphylococcus aureus 
Molecular microbiology  2013;90(4):734-743.
Staphylococcus aureus encodes the specialized ESAT-6 Secretion System (ESS). EsxA and EsxB are secreted by the ESS pathway, and share sequence features of ESAT-6 and CFP-10 of the Type VII Secretion System (T7SS) of Mycobacterium tuberculosis. Unlike ESAT-6 and CFP-10, EsxA and EsxB do not interact. Instead, EsxB associates with a novel substrate, EsxD, and EsxA dimerizes with itself or EsxC (EsaC). Unlike EsxA and EsxB, EsxC and EsxD do not share obvious sequence features of WXG100 proteins nor PE/PPE and Esp families of proteins, all of which belong to the pfam EsxAB clan of mycobacterial T7SS. EsxD carries the C terminal motif YxxxD/E that has been proposed to target T7 substrates for secretion in mycobacteria. Here, we find that deletion, but not amino acid substitutions, in this motif prevent secretion of EsxA and EsxC but not EsxB or EsxD. This is unlike the genetic inactivation of esxA, esxB, esxC or esxD that leads to loss of secretion of all four substrates. Thus, substrate secretion can be uncoupled by deleting the last six amino acids of EsxD. The physical association of EsxC and EsxD with canonical WXG100 proteins suggests that these proteins belong to the EsxAB clan.
PMCID: PMC3951145  PMID: 24033479
6.  Lineage tracing and cell ablation identifies a post-Aire expressing thymic epithelial cell population 
Cell reports  2013;5(1):10.1016/j.celrep.2013.08.038.
Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and MHC II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire+ mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire− mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates towards the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.
PMCID: PMC3820422  PMID: 24095736
7.  Oxidized CaMKII Triggers Atrial Fibrillation 
Circulation  2013;128(16):10.1161/CIRCULATIONAHA.113.003313.
Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF.
Methods and Results
We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion.
Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.
PMCID: PMC3876034  PMID: 24030498
atrial fibrillation; arrhythmia mechanisms; calcium/calmodulin-dependent protein kinase II; angiotensin II; reactive oxygen species
8.  Critical roles of junctophilin-2 in T-tubule and excitation–contraction coupling maturation during postnatal development 
Cardiovascular Research  2013;100(1):54-62.
Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation.
Methods and results
Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TTpower) index revealed a progressive increase in TTpower between postnatal Days 10 and 19. By postnatal Day 19, TTpower was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TTpower. We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca2+ transient amplitude and marked increase in Ca2+ release dyssynchrony, Ca2+ alternans, and spontaneous Ca2+ waves, leading to contractile failure.
Our data identify a critical role for JP2 in T-tubule and excitation–contraction coupling maturation during development.
PMCID: PMC3778961  PMID: 23860812
T-tubules; Junctophilin-2; Excitation–contraction coupling; Calcium; Heart development
9.  Extrathymic Aire-Expressing Cells are a Distinct Bone Marrow-Derived Population that Induce Functional Inactivation of CD4+ T Cells 
Immunity  2013;39(3):560-572.
The autoimmune regulator (AIRE) is essential for prevention of autoimmunity; its role is best understood in the thymus where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)hi, CD80lo, CD86lo, epithelial cell adhesion molecule (EpCAM)hi, CD45lo bone marrow-derived peripheral antigen presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4+ T cells through a mechanism that does not require regulatory T cells (Treg), and is resistant to innate inflammatory stimuli. Together these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.
PMCID: PMC3804105  PMID: 23993652
10.  Insights into type 1 diabetes from the autoimmune polyendocrine syndromes 
Purpose of review:
Advances in human genetics and investigations in animal models of autoimmune disease have allowed insight into the basic mechanisms of immunologic tolerance. These advances allow us to understand the pathogenesis of Type 1 diabetes and other autoimmune diseases as never before. Here, we discuss the tolerance mechanisms of the autoimmune polyendocrine syndromes and their relevance to Type 1 diabetes.
Recent findings:
Defects in central tolerance with alteration of self-antigen expression levels in the thymus are a potent cause of autoimmunity. Peripheral tolerance defects that alter T cell activation and signaling also play an important role in the pathogenesis of diabetes and other associated autoimmune disorders, with multiple modest defects working in concert to produce disease. Regulation of the immune response through the action of regulatory T cells is a potent mode of tolerance induction in autoimmunity that is important in Type 1 diabetes.
Rare syndromes of autoimmunity provide a valuable window into the breakdown of tolerance and identify multiple checkpoints that are critical for generation of autoimmunity. Understanding the application of these in Type 1 diabetes will allow the development of future immunomodulatory therapies in the treatment and prevention of disease.
PMCID: PMC4165040  PMID: 23770732
AIRE; IPEX; immune tolerance; polyglandular autoimmunity
11.  Why has it taken so long to learn what we still don’t know? 
Circulation research  2013;113(7):10.1161/CIRCRESAHA.113.302137.
PMCID: PMC3871173  PMID: 24030016
CaV1.2; phosphorylation; protein kinases
12.  Complications associated with tibial plateau leveling osteotomy: A retrospective of 1519 procedures 
The Canadian Veterinary Journal  2014;55(3):249-254.
This retrospective study identified complications associated with tibial plateau leveling osteotomy (TPLO) and predisposing factors for these complications in a large population of dogs from a metropolitan area with cruciate ligament deficiency. There were 943 dogs that underwent unilateral TPLO and 288 with staged bilateral TPLO for a total of 1519 procedures. There were 47 cases with at least 1 major complication and 126 cases with at least 1 minor complication but no major complications. The total complication rate (major or minor) was 11.4% [95% confidence interval (CI) estimate: 9.8%, 13.2%]; the major complication rate was 3.1% (95% CI: 2.3%, 4.1%); and the minor complication rate was 8.3% (95% CI: 7.0%, 9.8%). Factors associated with development of complications included being a German shepherd dog [odds ratio (OR): 3.2], tibial plateau angle > 30° (OR: 1.6), and heavier weights (for every 4.5 kg increase in body weight the OR increased by 1.10). Tibial plateau leveling osteotomy is a common treatment for dogs with cruciate ligament deficiency and has a low complication rate.
PMCID: PMC3923481  PMID: 24587508
13.  Ca2+ Cycling in Heart Failure 
Circulation research  2013;113(6):690-708.
Ca2+ plays a crucial role in connecting membrane excitability with contraction in myocardium. The hallmark features of heart failure are mechanical dysfunction and arrhythmias; defective intracellular Ca2+ homeostasis is a central cause of contractile dysfunction and arrhythmias in failing myocardium. Defective Ca2+ homeostasis in heart failure can result from pathological alteration in the expression and activity of an increasingly understood collection of Ca2+ homeostatic binding proteins, ion channels and enzymes. This review focuses on the molecular mechanisms of defective Ca2+ cycling in heart failure and consider how fundamental understanding of these pathways may translate into novel and innovative therapies.
PMCID: PMC4080816  PMID: 23989713
Calcium; heart failure; excitation-contraction coupling; CaMKII; mitochondria
14.  Evaluation of Resettin® on serum hormone levels in sedentary males 
Comparisons of hormones such as dihydrotestosterone (DHT), estradiol (E2), and testosterone indicate their impact on metabolism and body composition. While less is known regarding DHT and E2, testosterone is an androgenic metabolic hormone capable of positively regulating a variety of anabolic and androgenic processes in the body. Accordingly, it has been postulated that the age-related reduction in serum testosterone levels leads to reductions in lean muscle mass, bone mineral density, and other physical conditions that impair physical performance and decrease quality of life. Preliminary studies suggest that key ingredients found in Resettin®/MyTosterone™, a natural supplement containing the carotenoid astaxanthin from Haematococcus pluvialis and Saw Palmetto berry lipid extract from Serenoa repens, could positively impact testosterone levels. To investigate the clinical efficacy of Resettin®, the serum profiles of testosterone, E2 and DHT in healthy sedentary males before and after Resettin® treatment were evaluated in a randomized, placebo controlled clinical trial.
Twenty healthy, sedentary men between the ages of 21 and 70 were randomized into either an 800 mg/day or 1200 mg/day Resettin®/MyTosterone™ treatment group or lecithin, which was used as the placebo. After a 14-day treatment period, there was a 14-day washout period. After the wash-out period, participants were crossed over within their respective group to either Resettin®/MyTosterone™ or the lecithin placebo for 14 days.
After 14 days, participants receiving 800 mg per day of Resettin® had significantly reduced baseline-subtracted serum DHT levels in comparison to the placebo control group. While after 14 days, participants receiving 1200 mg per day of Resettin® had significantly reduced baseline-subtracted serum DHT and E2 levels in comparison to the placebo control group. Moreover, participants receiving 1200 mg per day of Resettin® experienced a 38% increase in serum testosterone levels in comparison to the placebo control group, but the effect did not reach statistical significance.
Although additional studies will be required to evaluate how Resettin® may promote proper testosterone regulation, these findings indicate that Resettin® can favorably influence serum hormone profiles in men.
PMCID: PMC4151021  PMID: 25183955
Testosterone; Estradiol; Dihydrotestosterone; 5-alpha-reductase; Aromatase inhibitor; Resistance training; Sarcopenia
15.  Directed cardiomyogenesis of autologous human induced pluripotent stem cells recruited to infarcted myocardium with bioengineered antibodies 
Myocardial infarctions constitute a major factor contributing to non-natural mortality world-wide. Clinical trials of myocardial regenerative therapy, currently pursued by cardiac surgeons, involve administration of stem cells into the hearts of patients suffering from myocardial infarctions. Unfortunately, surgical acquisition of these cells from bone marrow or heart is traumatic, retention of these cells to sites of therapeutic interventions is low, and directed differentiation of these cells in situ into cardiomyocytes is difficult. The specific aims of this work were: (1) to generate autologous, human, pluripotent, induced stem cells (ahiPSCs) from the peripheral blood of the patients suffering myocardial infarctions; (2) to bioengineer heterospecific antibodies (htAbs) and use them for recruitment of the ahiPSCs to infarcted myocardium; (3) to initiate in situ directed cardiomyogenesis of the ahiPSCs retained to infarcted myocardium.
Peripheral blood was drawn from six patients scheduled for heart transplants. Mononuclear cells were isolated and reprogrammed, with plasmids carrying six genes (NANOG, POU5F1, SOX2, KLF4, LIN28A, MYC), to yield the ahiPSCs. Cardiac tissues were excised from the injured hearts of the patients, who received transplants during orthotopic surgery. These tissues were used to prepare in vitro models of stem cell therapy of infarcted myocardium. The htAbs were bioengineered, which simultaneously targeted receptors displayed on pluripotent stem cells (SSEA-4, SSEA-3, TRA-1-60, TRA-1-81) and proteins of myocardial sarcomeres (myosin, α-actinin, actin, titin). They were used to bridge the ahiPSCs to the infarcted myocardium. The retained ahiPSCs were directed with bone morphogenetic proteins and nicotinamides to differentiate towards myocardial lineage.
The patients’ mononuclear cells were efficiently reprogrammed into the ahiPSCs. These ahiPSCs were administered to infarcted myocardium in in vitro models. They were recruited to and retained at the treated myocardium with higher efficacy and specificity, if were preceded with the htAbs, than with isotype antibodies or plain buffers. The retained cells differentiated into cardiomyocytes.
The proof of concept has been attained, for reprogramming the patients’ blood mononuclear cells (PBMCs) into the ahiPSCs, recruiting these cells to infarcted myocardium, and initiating their cardiomyogenesis. This novel strategy is ready to support the ongoing clinical trials aimed at regeneration of infarcted myocardium.
PMCID: PMC4131312  PMID: 25132967
Myocardial infarction; cardiac regeneration; stem cell therapy; recruitment and retention of stem cells; autologous human induced pluripotent stem cell; heterospecific, tetravalent antibodies; stage specific embryonic antigen; tumor related antigen; pluripotent stem cell tumorigenicity
16.  Inhibition of CaMKII Does Not Attenuate Cardiac Hypertrophy in Mice with Dysfunctional Ryanodine Receptor 
PLoS ONE  2014;9(8):e104338.
In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca2+ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2ADA/ADA mice.
PMCID: PMC4122402  PMID: 25093823
17.  Differences in Tibia Morphology between the Sound and Affected Sides in Ankle Foot Orthosis using Survivors of Stroke 
To examine differences between the affected and sound limbs in ankle-foot orthosis (AFO)-using survivors of stroke. AFOs and gait aids are commonly used to allow survivors of stroke to ambulate. Previous investigations of bone mineral density (BMD) in stroke survivors cite gait aid use, but have not controlled for their use when presenting outcomes.
Observational study.
University of Oklahoma Bone Density laboratory.
Nine ambulatory, AFO-using survivors of stroke (ages 55-74 years, 13.5±4.4 years post-stroke, 6.5±1.4 years of AFO use).
Not applicable.
Main Outcome Measures
Total body and hip areal BMD (aBMD) and bone mineral content (BMC) were assessed by DXA. The 4%, 38% and 66% sites of both tibiae were measured with peripheral Quantitative Computed Tomography (pQCT) for total, cortical and trabecular volumetric BMD (vBMD) and BMC. Bone geometry, bone strength index (BSI), strength strain index (SSI) and moments of inertia (Imin, Imax) were determined.
Total hip and trochanter BMC and aBMD were 7%-19% greater on the sound side (p<0.05). Total BMC and vBMD were 2%-21% (p<0.05) on the sound limb, depending on site. Trabecular BMC and vBMD and BSI values were 19%, 21%, and 31% higher (p<0.05) on sound limb at the 4% site. Cortical BMC and vBMD (p<0.05), and cortical thickness (p<0.01) were greater on the sound side at the 38% and 66% sites. Cortical area and bone strength (SSI, Imin) were greater (p<0.05) at the 66% site. Endosteal circumferences were greater on the affected side (p<0.01).
Interlimb differences in bone characteristics following a stroke persist despite returning to ambulatory status with AFO use.
PMCID: PMC4119800  PMID: 23123505
tibia; stroke; orthosis; osteoporosis
18.  Differential Control of Calcium Homeostasis and Vascular Reactivity by CaMKII 
Hypertension  2013;62(2):10.1161/HYPERTENSIONAHA.113.01508.
The multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca2+ channel (LTCC) current (ICa), cytoplasmic and sarcoplasmic reticulum (SR) Ca2+ and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual ICa 50 ms after peak amplitude, indicative of loss of LTCC-dependent ICa facilitation. Treatment with angiotensin-II or phenylephrine increased the intracellular Ca2+ concentration ([Ca2+]i) in WT but not TG SM-CaMKIIN VSMC. The difference in [Ca2+]i was abolished by pretreatment with nifedipine, an LTCC antagonist. In TG SM-CaMKIIN VSMC, the total SR Ca2+ content was reduced as a result of diminished SR Ca2+ ATPase (SERCA) activity via impaired derepression of the SERCA inhibitor phospholamban. Despite the differences in [Ca2+]i, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin-II and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.
PMCID: PMC3868483  PMID: 23753415
CaMKII; Ca2+ signaling; contraction; L-type Ca2+ channel
19.  Unique Presentation of Cerebellopontine Angle Choroid Plexus Papillomas: Case Report and Review of the Literature 
Objectives We present the case of a choroid plexus papilloma (CPP) in the cerebellopontine angle (CPA), describe the different appearances of CPPs with a variety of imaging techniques, and discuss the differential diagnosis of CPA tumors.
Participant and Design We report the case of a 52-year-old woman with headache, tinnitus, and unilateral hearing impairment whose preoperative magnetic resonance imaging revealed a heterogeneously enhancing CPA mass that extended into the internal auditory canal.
Main Outcome Measures, Results, and Conclusion The preoperative imaging appearance of the lesion was most consistent with that of a schwannoma. Postoperative histopathologic examination found the tumor to be a CPP with cuboidal epithelial cells overlying fibrovascular stroma. CPPs are rare benign central nervous system neoplasms arising from choroid plexus epithelium. The most common site of presentation is in the fourth ventricle in adults and the lateral ventricles in children. CPPs rarely occur in the CPA, and when they do, clinical-radiologic diagnosis is difficult due to both the rarity of this presentation and to nonspecific radiological features.
PMCID: PMC4110152  PMID: 25083384
cerebellopontine angle; choroid plexus papilloma
20.  Recruitment and retention of human autologous CD34+ CD117+ CD133+ bone marrow stem cells to infarcted myocardium followed by directed vasculogenesis: Novel strategy for cardiac regeneration § 
Ongoing clinical trials, in regenerative therapy of patients suffering from myocardial infarctions, rely primarily upon administration of bone marrow stem cells to the infarcted zones. Unfortunately, low retention of these cells, to the therapeutic delivery sites, reduces effectiveness of this strategy; thus it has been identified as the most critical problem for advancement of cardiac regenerative medicine.
Specific aims
The specific aim of this work was three-fold: (1) to isolate highly viable populations of human, autologous CD34+, CD117+, and CD133+ bone marrow stem cells; (2) to bioengineer heterospecific, tetravalent antibodies and to use them for recruiting of the stem cells to regenerated zones of infarcted myocardium; (3) to direct vasculogenesis of the retained stem cells with the defined factors.
Patients. Methods.
Cardiac tissue was biopsied from the hearts of the patients, who were receiving orthotopic heart transplants after multiple cardiac infarctions. This tissue was used to engineer fully human in vitro models of infarcted myocardium. Bone marrow was acquired from these patients. The marrow cells were sorted into populations of cells displaying CD34, CD117, and CD133. Heterospecific, tetravalent antibodies were bioengineered to bridge CD34, CD117, CD133 displayed on the stem cells with cardiac myosin of the infarcted myocardium. The sorted stem cells were administered to the infarcted myocardium in the in vitro models.
Administration of the bioengineered, heterospecific antibodies preceding administration of the stem cells greatly improved the stem cells’ recruitment and retention to the infarcted myocardium. Treatment of the retained stem cells with vascular endothelial growth factor and angiopoietin efficiently directed their differentiation into endothelial cells, which expressed vascular endothelial cadherin, platelet / endothelial cell adhesion molecule, claudin, and occludin, while forming tight and adherens junctions.
This novel strategy improved retention of the patients’ autologous bone marrow stem cells to the infarcted myocardium followed by directed vasculogenesis. Therefore, it is worth pursuing it in support of the ongoing clinical trials of cardiac regenerative therapy.
PMCID: PMC4100620  PMID: 25045527
Myocardial infarction; Regenerative medicine; Bone marrow stem cells; Stem cell therapy; Vasculogenesis; Heterospecific tetravalent antibodies
21.  Age and sex differences in tibia morphology in healthy adult Caucasians 
Bone  2012;50(6):1324-1331.
Variability in peripheral Quantitative Computed Tomography (pQCT) measurement sites limits direct comparisons of results between studies. Further, it is unclear what estimates of bone strength are most indicative of changes due to aging, disease, or interventions. The purpose of this study was to examine age group and sex differences in tibia morphology. Additional purposes of this study were to determine which tibia site or sites are most sensitive for detecting age and sex differences.
Self-identifying Caucasian men (n=55) and women (n=59) ages 20-59 years and separated by decades had their non-dominant tibias measured with pQCT (Stratec XCT 3000) at every 10% of the limb length from 5%-85% (distal to proximal). Volumetric BMD and BMC of the total, cortical and trabecular bone were determined, as well as periosteal (PeriC) and endosteal (EndoC) circumferences, and cortical thickness (CTh).
There were significant (p<0.01) site effects for all BMC, vBMD, PeriC and EndoC measures. Large sex differences (men>women) in Tot.BMC (21-28%) were paralleled by differences in Cort.BMC (21-25%) (p<0.01). Site*sex interaction effects were significant (p<0.05) for BMC (peak sex difference: 5%, 15%, 25%, 85% sites) and circumference (peak sex difference: 65% site) variables. CTh and total vBMD were lowest (p<0.05) in 50-59 yr group, and EndoC was highest in the 50-59 yr group. Site*age interactions existed for Cort.vBMD, Tot.BMC (85% site), and EndoC (25%, 35%, 55%-85% sites). Correcting for bone free lean body mass (BFLBM) greatly reduced sex differences, eliminating sex*site interaction effects, but sex main effects remained significant. Correcting for BFLBM did not eliminate age effects.
The magnitude of age and sex differences in tibia variables varied by measurement site demonstrating the need for standardization of measurement sites.
PMCID: PMC4082662  PMID: 22449446
pQCT; morphology; lean mass; vBMD
23.  Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads 
The Journal of General Physiology  2014;143(1):119-134.
ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle–specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.
PMCID: PMC3874572  PMID: 24344248
25.  Mitochondrial Calcium Uniporter Activity Is Dispensable for MDA-MB-231 Breast Carcinoma Cell Survival 
PLoS ONE  2014;9(5):e96866.
Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.
PMCID: PMC4011874  PMID: 24802861

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