The prognosis of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is poor. Chemotherapy is rarely curative and tyrosine kinase inhibitors (TKIs) induce only transient responses. Heat shock protein 90 (Hsp90) is a chaperone protein that is important in signal transduction, cell cycle control, and transcription regulation in both normal and leukemia cells. In the current study, we tested the growth inhibitory and apoptotic effects of a novel Hsp90 inhibitor, EC141 on the Ph+ ALL lines Z-119, Z-181, and Z-33, as well as primary bone marrow-derived blasts from patients with newly diagnosed Ph+ ALL. We found that EC141 inhibited the growth of Ph+ ALL cells in a concentration-dependent manner with IC50 ranged from 1 to 10 nM. EC141 also inhibited the proliferation of primary bone marrow-derived blasts using the ALL blast colony assay. EC141 down-regulated Hsp90 and up-regulated Hsp70 protein levels, inhibited CrkL phosphorylation, and induced degradation of Bcr-Abl p190 protein through ubiquitin-dependent proteasomal pathway. Furthermore, exposure of Ph+ ALL cells to EC141 resulted in activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and induction of apoptosis. In conclusion, our data suggest that EC141 is a potent Hsp90 inhibitor with activity against Ph+ ALL. Further studies to investigate the anticancer effect of EC141 either as a single agent, or in combination in Ph+ ALL and other hematological malignancies are warranted.
Leukemia; Hsp90 inhibitor; EC141; Apoptosis; Ph+ ALL
Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells.
Routine clinical use of novel free-breathing, motion-corrected, averaged late gadolinium enhancement (moco-LGE) cardiovascular magnetic resonance may have advantages over conventional breath held LGE (bh-LGE) especially in vulnerable patients.
Methods and Results
In 390 consecutive patients, we collected bh-LGE and moco-LGE with identical image matrix parameters. In 41 patients, bh-LGE was abandoned due to image quality issues, including 10 with myocardial infarction (MI). When both were acquired, MI detection was similar (McNemar test, p=0.4) with high agreement (kappa statistic 0.95). With artifact-free bh-LGE images, pixelwise MI measures correlated highly (R2=0.96) without bias. Moco-LGE was faster, and image quality and diagnostic confidence were higher on blinded review (p<0.001 for all). Over a median of 1.2 years, 20 heart failure hospitalizations and 18 deaths occurred. For bh-LGE, but not moco-LGE, inferior image quality and bh-LGE non acquisition were linked to patient vulnerability confirmed by adverse outcomes (logrank p<0.001). Moco-LGE significantly stratified risk on the full cohort (logrank p<0.001), but bh-LGE did not (logrank p=0.056) since a significant number of vulnerable patients did not receive bh-LGE (due to arrhythmia or inability to breath hold).
MI detection and quantification are similar between moco-LGE and bh-LGE when bh-LGE can be acquired well, but bh-LGE quality deteriorates with patient vulnerability. Acquisition time, image quality, diagnostic confidence and the number of successfully scanned patients are superior with moco-LGE which extends LGE-based risk stratification to include patients with vulnerability confirmed by outcomes. Moco-LGE may be suitable for routine clinical use.
myocardial delayed enhancement; magnetic resonance imaging; myocardial infarction
Approximately 1,000 microRNAs (miRs) are present in the human genome; however, little is known about the regulation of miR transcription. Because miR levels are deregulated in chronic lymphocytic leukemia (CLL) and signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL, we sought to determine whether STAT3 affects the transcription of miR genes in CLL cells.
We used publically available data from the ENCODE project to identify putative STAT3 binding sites in the promoters of miR genes. Then we transfected CLL cells with STAT3-shRNA or with an empty vector, and to determine which miRs are differentially expressed, we used a miR microarray approach followed by validation of the microarray results for 6 miRs using quantitative real-time polymerase chain reaction (qRT-PCR).
We identified putative STAT3 binding sites in 160 promoter regions of 200 miRs, including miR-21, miR-29, and miR-155, whose levels have been reported to be upregulated in CLL. Levels of 72 miRs were downregulated (n = 63) or upregulated (n = 9). qRT-PCR confirmed the array data in 5 of 6 miRs.
The presence of activated STAT3 has a profound effect on miR expression in CLL cells.
CLL; microRNA; STAT3
Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca2+) is implicated in hypertrophic signaling pathways, the specific role of Ca2+ influx through the L-type Ca2+ channel (ICa-L) has been controversial and is the topic of this study. To determine if and how sustained increases in ICa-L induces PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca2+ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP.
In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume and cross sectional area and the expression of PCH markers in a time- and dose- dependent manner. PCH was associated with a hypercontractile phenotype including enhanced ICa-L, fractional shortening, peak Ca2+ transient, at the myocyte level, greater ejection fraction and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased ICa-L, cell volume, protein synthesis, NFAT and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of ICa-L, intracellular Ca2+, calcineurin, CaMK II and SERCA.
Increasing ICa-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved.
L-type calcium channel (or Cav1.2); β2a subunit; Ventricular Myocyte; hypertrophy; transgenic mouse
Activation of pro-survival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia-reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs) and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.
A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac β-adrenergic receptor (βAR) signaling. Chronic HF studies have been done with GRK2 knockout mice as well as expression of the βARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model.
Methods and Results
We demonstrate, using cardiac-specific GRK2 and βARKct expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with βARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2 overexpressing mice (45.0±2.8% vs. 31.3±2.3% in controls) and significantly smaller in βARKct mice (16.8±1.3%, p<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via βARKct and these anti-apoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of β2ARs abolished βARKct-mediated cardioprotection suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival.
The novel effect of reducing acute ischemic myocardial injury via increased Akt activity adds significantly to the therapeutic potential of GRK2 inhibition with the βARKct to not only chronic HF but also potentially in acute ischemic injury conditions.
acute myocardial ischemia; ischemia/reperfusion injury; cardioprotection; G protein-coupled receptor kinase-2; βARKct; Myocyte apoptosis
Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study.
We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca2+-handling defects could avert post-MI cardiac pump dysfunction.
Methods and Results
MI was produced in mice with inducible, cardiac-specific expression of the β2a subunit of the L-type Ca2+ channel. Myocyte and cardiac function were compared in control and β2a animals before and after MI. β2a myocytes had increased Ca2+ current; sarcoplasmic reticulum Ca2+ load, contraction and Ca2+ transients (versus controls), and β2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in β2a versus control hearts after MI. β2a animals also had poorer survival after MI. Myocytes isolated from β2a hearts after MI did not develop depressed Ca2+ handling, and Ca2+ current, contractions, and Ca2+ transients were still above control levels (before MI).
Maintaining myocyte contractility after MI, by increasing Ca2+ influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.
myocardial infarction; cardiac contractility; heart failure; Ca2+ handling
Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.
Abnormal L-type Ca2+ channel (LTCC, also named Cav1.2) density and regulation are important contributors to depressed contractility in failing hearts. The LTCC agonist BAY K 8644 (BAY K) has reduced inotropic effects on failing myocardium. We hypothesized that BAY K effects on the LTCC current (ICaL) in failing myocytes would be reduced because of increased basal activity. Since support of the failing heart with a left ventricular assist device (LVAD) improves contractility and adrenergic responses, we further hypothesized that BAY K effects on ICaL would be restored in LVAD-supported failing hearts. We tested our hypotheses in human ventricular myocytes (HVMs) isolated from nonfailing (NF), failing (F), and LVAD-supported failing hearts. We found that 1) BAY K had smaller effects on ICaL in F HVMs compared with NF HVMs; 2) BAY K had diminished effects on ICaL in NF HVM pretreated with isoproterenol (Iso) or dibutyryl cyclic AMP (DBcAMP); 3) BAY K effects on ICaL in F HVMs pretreated with acetylcholine (ACh) were normalized; 4) Iso had no effect on NF HVMs pretreated with BAY K; 5) BAY K effects on ICaL in LVAD HVMs were similar to those in NF HVMs; 6) BAY K effects were reduced in LVAD HVMs pretreated with Iso or DBcAMP; 7) Iso had no effect on ICaL in LVAD HVMs pretreated with BAY K. Collectively, these results suggest that the decreased BAY K effects on LTCC in F HVMs are caused by increased basal channel activity, which should contribute to abnormal contractility reserve.
heart failure; L-type calcium channel; left ventricular assist device
Understanding binaural perception requires detailed analyses of the neural circuitry responsible for the computation of interaural time differences (ITDs). In the avian brainstem, this circuit consists of internal axonal delay lines innervating an array of coincidence detector neurons that encode external ITDs. Nucleus magnocellularis (NM) neurons project to the dorsal dendritic field of the ipsilateral nucleus laminaris (NL) and to the ventral field of the contralateral NL. Contralateral-projecting axons form a delay line system along a band of NL neurons. Binaural acoustic signals in the form of phase-locked action potentials from NM cells arrive at NL and establish a topographic map of sound source location along the azimuth. These pathways are assumed to represent a circuit similar to the Jeffress model of sound localization, establishing a place code along an isofrequency contour of NL. Three-dimensional measurements of axon lengths reveal major discrepancies with the current model; the temporal offset based on conduction length alone makes encoding of physiological ITDs impossible. However, axon diameter and distances between Nodes of Ranvier also influence signal propagation times along an axon. Our measurements of these parameters reveal that diameter and internode distance can compensate for the temporal offset inferred from axon lengths alone. Together with other recent studies these unexpected results should inspire new thinking on the cellular biology, evolution and plasticity of the circuitry underlying low frequency sound localization in both birds and mammals.
Sound; Localization; Auditory; Brainstem; Axon; Conduction; Velocity
Hypertension occurs with higher prevalence and morbidity in black Americans compared to other groups. Alterations in the signal transduction pathways of seven-transmembrane spanning receptors are found in hypertensive patients. GRKs play an important role in regulating this receptor signaling. The two most abundantly expressed GRKs in the cardiovascular system are GRK2 and GRK5 and each have unique substrates. Understanding changes in expression may give us insight into activated receptors in the pathophysiological progression of hypertension. In heart failure and Caucasian hypertensives, increased GRK2 expression arises because of neurohormal stimulation of particular receptors. GRK2 subsequently desensitizes specific receptors including β-adrenergic receptors. In blood pressure control, β-adrenergic receptor desensitization could lead to increased blood pressure. GRK2 and GRK5 mRNA were evaluated in lymphocytes of black Americans via quantitative real-time PCR. GRK2 mRNA expression directly correlated with systolic blood pressure and norepinephrine levels. GRK2 was elevated >30% among those with systolic blood pressure equal to or greater than 130mmHg. No significant correlation between GRK5 mRNA expression and blood pressure or catecholamines was observed. Diabetic status, age, sex and body mass index (BMI) were also examined compared to GRK2 expression using univariate and multi-variate analyses. GRK2 protein expression was elevated 2-fold in subjects with higher blood pressure and GRK activity was increased >40%. Our data suggest that GRK2, but not GRK5, is correlated with increasing blood pressure in black Americans. Understanding the receptors stimulated by increased neurohormonal activation may give insight into the pathophysiology of hypertension in this at-risk population.
hypertension; beta adrenergic receptors; lymphocytes; beta adrenergic receptor kinase; G protein-coupled receptors; catecholamines
Chronic ventricular pressure overload states, such as hypertension, and elevated levels of neurohormones (norepinephrine, angiotensin II, endothelin-1) initiate cardiac hypertrophy and dysfunction and share the property of being able to bind to Gq-coupled 7-transmembrane receptors. The goal of the current study was to determine the role of endogenous cardiac myocyte Gq signaling and its role in cardiac hypertrophy and dysfunction during high blood pressure (BP). We induced renal artery stenosis for 8 weeks in control mice and mice expressing a peptide inhibitor of Gq signaling (GqI) using a 2 kidney, 1 clip renal artery stenosis model. 8 weeks following chronic high BP, control mice had cardiac hypertrophy and depressed function. Inhibition of cardiomyocyte Gq signaling did not reverse cardiac hypertrophy but attenuated increases in a profile of cardiac profibrotic genes and genes associated with remodeling. Inhibition of Gq signaling also attenuated the loss of cardiac function. We determined that Gq signaling downstream of angiotensin II receptor stimulation negatively impacted β-adrenergic receptor (AR) responses and inhibition of Gq signaling was sufficient to restore βAR-mediated responses. Therefore, in this study we found that Gq signaling negatively impacts cardiac function during high BP. Specifically, we found that inhibition of AT1-Gq signaling augmented βAR mediated effects in a renal artery stenosis model of hypertension. These observations may underlie additional, beneficial effects of angiotensinogen converting enzyme (ACE) inhibitors and angiotensin receptor antagonists observed during times of hemodynamic stress.
receptors; adrenergic; beta; hypertension; signal transduction; myocytes; hypertrophy; cell signaling/signal transduction; genetically altered mice; heart failure - basic studies; hypertension - basic studies; Goldblatt model of hypertension
Devices for visualizing blood vessels have evolved from a rigid, illuminated tube (1913), to a tube with an added convex lens (1922), to one with a transparent inflatable balloon for displacing blood from the line of vision (1943), to a flexible angioscope (1960s). Recent fiberoptic developments make it possible to visualize the orifices of the coronary arteries and simultaneous laser angioplasty. The characteristic fluorescence of hematoporphyrin derivative under ultraviolet light has been visualized angioscopically in experimental atherosclerotic plaque, where it accumulates and acts as a marker.
However, several requirements need to be met in order for angioscopy to fulfill its therapeutic possibilities in angioplasty, thrombolytic therapy, intraoperative inspection of vascular anastomoses, and its diagnostic potential in distinguishing plaques from clots and pulmonary embolisms from other obstructions. These requirements are: (1) variously-sized angioscopes to accommodate iliac, femoral, renal, and coronary arteries; (2) percutaneous introducers in the various sizes to prevent back-bleeding; (3) a more flexible, easily manipulated fiberoptic; (4) a sufficiently inflatable balloon tip; (5) cross hairs and reference points in the optical system; and (6) optimal focal lengths for the areas to be visualized. Texas Heart Institute Journal 1986; 13:281-289)
Angiography; angioscopy; fiberoptic developments