Mesenchymal stem cells-derived extracellular vesicles for therapeutics of renal tuberculosis.

Extrapulmonary tuberculosis with a renal involvement can be a manifestation of a disseminated infection that requires therapeutic intervention, particularly with a decrease in efficacy of conventional regimens. In the present study, we investigated the therapeutic potency of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in the complex anti-tuberculosis treatment (ATT). A rabbit model of renal tuberculosis (rTB) was constructed by injecting of the standard strain Mycobacterium tuberculosis H37Rv into the cortical layer of the kidney parenchyma. Isolated rabbit MSC-EVs were intravenously administered once as an addition to standard ATT (isoniazid, pyrazinamide, and ethambutol). The therapeutic efficacy was assessed by analyzing changes of blood biochemical biomarkers and levels of anti- and pro-inflammatory cytokines as well as by renal computed tomography with subsequent histological and morphometric examination. The therapeutic effect of therapy with MSC-EVs was shown by ELISA method that confirmed a statistically significant increase of the anti-inflammatory and decrease of pro-inflammatory cytokines as compared to conventional treatment. In addition, there is a positive trend in increase of ALP level, animal weigh, and normalization of ADA activity that can indicate an improvement of kidney state. A significant reduction of the area of specific and interstitial inflammation indicated positive affect of MSC-EVs that suggests a shorter duration of ATT. The number of MSC-EVs proteins (as identified by mass-spectometry analysis) with anti-microbial, anti-inflammatory and immunoregulatory functions reduced the level of the inflammatory response and the severity of kidney damage (further proved by morphometric analysis). In conclusion, MSC-EVs can be a promising tool for the complex treatment of various infectious diseases, in particularly rTB.

Comparison of Autografts and Biodegradable 3D-Printed Composite Scaffolds with Osteoconductive Properties for Tissue Regeneration in Bone Tuberculosis.

Tuberculosis remains one of the major health problems worldwide. Besides the lungs, tuberculosis affects other organs, including bones and joints. In the case of bone tuberculosis, current treatment protocols include necrectomy in combination with conventional anti-tuberculosis therapy, followed by reconstruction of the resulting bone defects. In this study, we compared autografting and implantation with a biodegradable composite scaffold for bone-defect regeneration in a tuberculosis rabbit model. Porous three-dimensional composite materials were prepared by 3D printing and consisted of poly(ε-caprolactone) filled with nanocrystalline cellulose modified with poly(glutamic acid). In addition, rabbit mesenchymal stem cells were adhered to the surface of the composite scaffolds. The developed tuberculosis model was verified by immunological subcutaneous test, real-time polymerase chain reaction, biochemical markers and histomorphological study. Infected animals were randomly divided into three groups, representing the infection control and two experimental groups subjected to necrectomy, anti-tuberculosis treatment, and plastic surgery using autografts or 3D-composite scaffolds. The lifetime observation of the experimental animals and analysis of various biochemical markers at different time periods allowed the comparison of the state of the animals between the groups. Micro-computed tomography and histomorphological analysis enabled the evaluation of osteogenesis, inflammation and cellular changes between the groups, respectively.

The Use of Mesenchymal Stem Cells in the Complex Treatment of Kidney Tuberculosis (Experimental Study).

In recent years, the application of mesenchymal stem cells (MSCs) has been recognized as a promising method for treatment of different diseases associated with inflammation and sclerosis, which include nephrotuberculosis. The aim of our study is to investigate the effectiveness of MSCs in the complex therapy of experimental rabbit kidney tuberculosis and to evaluate the effect of cell therapy on the reparative processes. Methods: To simulate kidney tuberculosis, a suspension of the standard strain Mycobacterium tuberculosis H37Rv (106 CFU) was used, which was injected into the cortical layer of the lower pole parenchyma of the left kidney under ultrasound control in rabbits. Anti-tuberculosis therapy (aTBT) was started on the 18th day after infection. MSCs (5 × 107 cells) were transplanted intravenously after the start of aTBT. Results: 2.5 months after infection, all animals showed renal failure. Conducted aTBT significantly reduced the level of albumin, ceruloplasmin, elastase and the severity of disorders in the proteinase/inhibitor system and increased the productive nature of inflammation. A month after MSC transplantation, the level of inflammatory reaction activity proteins decreased, the area of specific and destructive inflammation in kidneys decreased and the formation of mature connective tissue was noted, which indicates the reparative reaction activation.

Clinical characteristics and outcome of infective endocarditis due to Abiotrophia and Granulicatella compared to Viridans group streptococci.

OBJECTIVE: To describe the clinical characteristics and outcome of Abiotrophia and Granulicatella infective endocarditis and compare them with Viridans group streptococci infective endocarditis. METHODS: All patients in the International Collaboration on Endocarditis (ICE) - prospective cohort study (PCS) and the ICE-PLUS cohort were included (n = 8112). Data from patients with definitive or possible IE due to Abiotrophia species, Granulicatella species and Viridans group streptococci was analyzed. A propensity score (PS) analysis comparing the ABI/GRA-IE and VGS-IE groups according to a 1:2 ratio was performed. RESULTS: Forty-eight (0.64%) cases of ABI/GRA-IE and 1,292 (17.2%) VGS-IE were included in the analysis. The median age of patients with ABI/GRA-IE was lower than VGS-IE (48.1 years vs. 57.9 years; p = 0.001). Clinical features and the rate of in-hospital surgery was similar between ABI/GRA-IE and VGS-IE (52.1% vs. 45.4%; p = 0.366). Unadjusted in-hospital death was lower in ABI/GRA-IE than VGS-IE (2.1% vs. 8.8%; p = 0.003), and cumulative six-month mortality was lower in ABI/GRA-IE than VGS-IE (2.1% vs. 11.9%; p<0.001). After PS analysis, in-hospital mortality was similar in both groups, but six-month mortality was lower in the ABI/GRA IE group (2.1% vs. 10.4%; p = 0.029). CONCLUSIONS: Patients with ABI/GRA-IE were younger, had similar clinical features and rates of surgery and better prognosis than VGS-IE.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Dual Activation of Phosphodiesterase 3 and 4 Regulates Basal Cardiac Pacemaker Function and Beyond.

The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca2+ releases (LCRs).

Urethroplasty with a bilayered poly-D,L-lactide-co-ε-caprolactone scaffold seeded with allogenic mesenchymal stem cells.

Reconstructive surgery for urethral defects employing tissue-engineered scaffolds represents an alternative treatment for urethroplasty. The aim of this study was to compare the therapeutic efficacy of the bilayer poly-D,L-lactide/poly-ε-caprolactone (PL-PC) scaffold seeded with allogenic mesenchymal stem cells (MSCs) for urethra reconstruction in a rabbit model with conventional urethroplasty employing an autologous buccal mucosa graft (BG). The inner layer of the scaffold based on poly-D,L-lactic acid (PL) was seeded with MSCs, while the outer layer, prepared from poly-ε-caprolactone, protected the surrounding tissues from urine. To track the MSCs in vivo, the latter were labeled with superparamagnetic iron oxide nanoparticles. In rabbits, a dorsal penile defect was reconstructed employing a BG or a PL-PC graft seeded with nanoparticle-labeled MSCs. In the 12-week follow-up period, no complications were detected. Subsequent histological analysis demonstrated biointegration of the PL-PC graft with surrounding urethral tissues. Less fibrosis and inflammatory cell infiltration were observed in the experimental group as compared with the BG group. Nanoparticle-labeled MSCs were detected in the urothelium and muscular layer, co-localizing with the urothelium cytokeratin marker AE1/AE3, indicating the possibility of MSC differentiation into neo-urothelium. Our results suggest that a bilayer MSCs-seeded scaffold could be efficiently employed for urethroplasty.

Dual Activation of Phosphodiesterases 3 and 4 Regulates Basal Spontaneous Beating Rate of Cardiac Pacemaker Cells: Role of Compartmentalization?

Spontaneous firing of sinoatrial (SA) node cells (SANCs) is regulated by cyclic adenosine monophosphate (cAMP)-mediated, protein kinase A (PKA)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from ryanodine receptors (RyR). The LCRs occur during diastolic depolarization (DD) and activate an inward Na+/Ca2+ exchange current that accelerates the DD rate prompting the next action potential (AP). Basal phosphodiesterases (PDEs) activation degrades cAMP, reduces basal cAMP/PKA-dependent phosphorylation, and suppresses normal spontaneous firing of SANCs. The cAMP-degrading PDE1, PDE3, and PDE4 represent major PDE activities in rabbit SANC, and PDE inhibition by 3-isobutyl-1-methylxanthine (IBMX) increases spontaneous firing of SANC by ∼50%. Though inhibition of single PDE1-PDE4 only moderately increases spontaneous SANC firing, dual PDE3 + PDE4 inhibition produces a synergistic effect hastening the spontaneous SANC beating rate by ∼50%. Here, we describe the expression and distribution of different PDE subtypes within rabbit SANCs, several specific targets (L-type Ca2+ channels and phospholamban) regulated by basal concurrent PDE3 + PDE4 activation, and critical importance of RyR Ca2+ releases for PDE-dependent regulation of spontaneous SANC firing. Colocalization of PDE3 and PDE4 beneath sarcolemma or in striated patterns inside SANCs strongly suggests that PDE-dependent regulation of cAMP/PKA signaling might be executed at the local level; this idea, however, requires further verification.

Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells Is Regulated by Dual Activation of PDEs (Phosphodiesterases) 3 and 4.

BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.

Application of the allogenic mesenchymal stem cells in the therapy of the bladder tuberculosis.

Urogenital tuberculosis (TB) often leads to contraction of the bladder, a reduction of the urinary reservoir capacity, and, in the latest stage, to real microcystitis up to full obliteration. Bladder TB Stage 4 is unsuitable for conservative therapy, and cystectomy with subsequent enteroplasty is indicated. In this study, using a model of bladder TB in New Zealand rabbits, the therapeutic efficacy of the interstitial injection of autologous bone-derived mesenchymal stem cells (MSCs) combined with standard anti-TB treatment in the restoration of the bladder function was demonstrated. For analysis of the MSC distribution in tissues, the latter were labelled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated the high intracellular incorporation of nanoparticles and the absence of cytotoxicity on MSC viability and proliferation. A single-dose administration of MSCs into the bladder mucosal layer significantly reduced the wall deformation and inflammation and hindered the development of fibrosis, which was proven by the subsequent histological assay. Confocal microscopy studies of the bladder cryosections confirmed the presence of superparamagnetic iron oxide nanoparticle-labelled MSCs in different bladder layers of the treated animals, thus indicating the role of stem cells in bladder regeneration.

Experimental bladder regeneration using a poly-l-lactide/silk fibroin scaffold seeded with nanoparticle-labeled allogenic bone marrow stromal cells.

In the present study, a poly-l-lactide/silk fibroin (PL-SF) bilayer scaffold seeded with allogenic bone marrow stromal cells (BMSCs) was investigated as a potential approach for bladder tissue engineering in a model of partial bladder wall cystectomy in rabbits. The inner porous layer of the scaffold produced from silk fibroin was designed to promote cell proliferation and the outer layer produced from poly-l-lactic acid to serve as a waterproof barrier. To compare the feasibility and efficacy of BMSC application in the reconstruction of bladder defects, 12 adult male rabbits were divided into experimental and control groups (six animals each) that received a scaffold seeded with BMSCs or an acellular one, respectively. For BMSC tracking in the graft in in vivo studies using magnetic resonance imaging, cells were labeled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated high intracellular incorporation of nanoparticles and the absence of a toxic influence on BMSC viability and proliferation. Following implantation of the graft with BMSCs into the bladder, we observed integration of the scaffold with surrounding bladder tissues (as detected by magnetic resonance imaging). During the follow-up period of 12 weeks, labeled BMSCs resided in the implanted scaffold. The functional activity of the reconstructed bladder was confirmed by electromyography. Subsequent histological assay demonstrated enhanced biointegrative properties of the PL-SF scaffold with cells in comparison to the control graft, as related to complete regeneration of the smooth muscle and urothelium tissues in the implant. Confocal microscopy studies confirmed the presence of the superparamagnetic iron oxide nanoparticle-labeled BMSCs in newly formed bladder layers, thus indicating the role of stem cells in bladder regeneration. The results of this study demonstrate that application of a PL-SF scaffold seeded with allogenic BMSCs can enhance biointegration of the graft in vivo and support bladder tissue regeneration and function.

hTERT and BIRC5 gene promoters for cancer gene therapy: A comparative study.

Human telomerase reverse transcriptase (hTERT) and survivin (BIRC5) gene promoters are frequently used for transcriptional targeting of tumor cells, yet there is no comprehensive comparative analysis allowing rational choice of a promoter for a particular therapy. In the current study, the transcriptional activity of hTERT, human BIRC5 and mouse Birc5 promoters and their modifications were compared in 10 human cancer cell lines using the luciferase reporter gene activity assay. The results revealed that BIRC5- and hTERT-based promoters had strikingly different cell specificities with comparable activities in only 40% of cell lines. Importantly, relative hTERT and BIRC5 transcript abundance cannot be used to predict the most potent promoter. Among the hTERT-based promoters that were assessed, modification with the minimal cytomegalovirus promoter generally resulted in the most potent activity. Mouse Birc5 and modified human BIRC5 promoters were superior to the unmodified human survivin promoter; however, their tumor specificities must be investigated further. In summary, the present results emphasize the desirability for construction of more universal tumor-specific promoters to efficiently target a wide spectrum of tumor cells.

Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design.

Basal phosphorylation of sarcoplasmic reticulum (SR) Ca(2+) proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca(2+) releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca(2+) sparks. We tested the hypothesis that in a physiologic Ca(2+) milieu, and independent of increased Ca(2+) influx, an increase in basal phosphorylation of SR Ca(2+) cycling proteins will convert stochastic Ca(2+) sparks into periodic, high-power Ca(2+) signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca(2+) release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca(2+)], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca(2+) releases of low power and low amplitude, as assessed by confocal Ca(2+) imaging and spectral analysis. When the kinetics of Ca(2+) pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, "clock-like" local Ca(2+) releases, partially synchronized in space and time (Ca(2+) wavelets), emerged, and the ensemble of these rhythmic local Ca(2+) wavelets generated a periodic high-amplitude Ca(2+) signal. Thus, a Ca(2+) clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca(2+) cycling increases and spontaneous local Ca(2+) release becomes partially synchronized. This unleashed Ca(2+) clock that emerges in a physiological Ca(2+) milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.

HACEK infective endocarditis: characteristics and outcomes from a large, multi-national cohort.

The HACEK organisms (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are rare causes of infective endocarditis (IE). The objective of this study is to describe the clinical characteristics and outcomes of patients with HACEK endocarditis (HE) in a large multi-national cohort. Patients hospitalized with definite or possible infective endocarditis by the International Collaboration on Endocarditis Prospective Cohort Study in 64 hospitals from 28 countries were included and characteristics of HE patients compared with IE due to other pathogens. Of 5591 patients enrolled, 77 (1.4%) had HE. HE was associated with a younger age (47 vs. 61 years; p<0.001), a higher prevalence of immunologic/vascular manifestations (32% vs. 20%; p<0.008) and stroke (25% vs. 17% p = 0.05) but a lower prevalence of congestive heart failure (15% vs. 30%; p = 0.004), death in-hospital (4% vs. 18%; p = 0.001) or after 1 year follow-up (6% vs. 20%; p = 0.01) than IE due to other pathogens (n = 5514). On multivariable analysis, stroke was associated with mitral valve vegetations (OR 3.60; CI 1.34-9.65; p<0.01) and younger age (OR 0.62; CI 0.49-0.90; p<0.01). The overall outcome of HE was excellent with the in-hospital mortality (4%) significantly better than for non-HE (18%; p<0.001). Prosthetic valve endocarditis was more common in HE (35%) than non-HE (24%). The outcome of prosthetic valve and native valve HE was excellent whether treated medically or with surgery. Current treatment is very successful for the management of both native valve prosthetic valve HE but further studies are needed to determine why HE has a predilection for younger people and to cause stroke. The small number of patients and observational design limit inferences on treatment strategies. Self selection of study sites limits epidemiological inferences.

LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

Subcellular trafficking and transfection efficacy of polyethylenimine-polyethylene glycol polyplex nanoparticles with a ligand to melanocortin receptor-1.

We have synthesized and investigated properties of new PEI-PEG-based polyplexes containing MC1SP-peptide, a ligand specific for melanocortin receptor-1 (targeted polyplexes), and control polyplexes without this ligand peptide (non-targeted polyplexes). The targeted polyplexes demonstrated receptor-mediated transfection of Cloudman S91 (clone M-3) murine melanoma cells that was more efficient than with the non-targeted ones. Transfection with the targeted polyplexes was inhibited by chlorpromazine, an inhibitor of the clathrin-mediated endocytosis pathway, and, to a lesser extent, by filipin III or nystatin, inhibitors of the lipid-raft endocytosis pathway, whereas transfection with the non-targeted polyplexes was inhibited mainly by nystatin or filipin III. The targeted polyplexes caused significantly higher in vivo transfection of melanoma tumor cells after intratumoral administration compared to the non-targeted control. The targeted polyplexes carrying the HSVtk gene, after ganciclovir administration, more efficiently inhibited melanoma tumor growth and prolonged the lifespan of DBA/2 tumor-bearing mice compared to the non-targeted ones. Packed targeted polyplexes appeared and accumulated in the melanoma cells 6h earlier than the non-targeted ones. The targeted polyplexes enter into the nuclei of the melanoma cells more rapidly than the non-targeted control, and this difference may also be attributed to processes of receptor-mediated endocytosis. We believe that these data may be useful for the optimization of polyplex systems.

A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling.

Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) clock," driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules ("surface membrane clock") to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca(2+) signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca(2+) cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca(2+) releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca(2+)]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by ß-adrenergic receptor (ß-AR) stimulation requires normal intracellular Ca(2+) cycling, because the ß-AR chronotropic effect is markedly blunted when SR Ca(2+) cycling is disrupted. Thus, AC-cAMP-PKA-Ca(2+) signaling cascade is a major mechanism of normal automaticity in mouse SANC.

Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP-mediated PKA-dependent Ca2+ cycling with surface membrane channels.

Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells to generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca(2+) and, specifically Ca(2+) release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca(2+) releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cell spontaneous firing. Local Ca(2+) releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca(2+) releases activate an inward Na(+)-Ca(2+) exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via beta-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca(2+) releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca(2+) cycling in regulation of the heart pacemaker clock, both in the basal state and during beta-adrenergic receptor stimulation.

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca(2+) cycling and I(KACh).

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G(i)-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I(f)) or L-type Ca(2+) currents (I(Ca,L)), respectively; and 2) direct G(i)-coupled activation of ACh-activated potassium current (I(KACh)). More recent studies, however, have indicated that Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was approximately 10 nM, half maximal inhibition (IC(50)) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G(i) inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I(f) blockade did not affect BRR at any [CCh] and that I(KACh) activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 50%. The dose response of BRR to CCh in the presence of I(KACh) blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC(50), CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G(i) activation and the extent of G(i) coupling to Ca(2+) cycling via PKA signaling or to I(KACh): at low [CCh], I(KACh) activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

The missing link in the mystery of normal automaticity of cardiac pacemaker cells.

Earlier studies of the initiating event of normal automaticity of the heart's pacemaker cells, inspired by classical quantitative membrane theory, focused upon ion currents (IK, I f) that determine the maximum diastolic potential and the early phase of the spontaneous diastolic depolarization (DD). These early DD events are caused by the prior action potential (AP) and essentially reflect a membrane recovery process. Events following the recovery process that ignite APs have not been recognized and remained a mystery until recently. These critical events are linked to rhythmic intracellular signals initiated by Ca2+ clock (i.e., sarcoplasmic reticulum [SR] cycling Ca2+). Sinoatrial cells, regardless of size, exhibit intense ryanodine receptor (RyR), Na+/Ca2+ exchange (NCX)-1, and SR Ca2+ ATPase-2 immunolabeling and dense submembrane NCX/RyR colocalization; Ca2+ clocks generate spontaneous stochastic but roughly periodic local subsarcolemmal Ca2+ releases (LCR). LCRs generate inward currents via NCX that exponentially accelerate the late DD. The timing and amplitude of LCR/I NCX-coupled events control the timing and amplitude of the nonlinear terminal DD and therefore ultimately control the chronotropic state by determining the timing of the I CaL activation that initiates the next AP. LCR period is precisely controlled by the kinetics of SR Ca2+ cycling, which, in turn, are regulated by 1) the status of protein kinase A-dependent phosphorylation of SR Ca2+ cycling proteins; and 2) membrane ion channels ensuring the Ca2+ homeostasis and therefore the Ca2+ available to Ca2+ clock. Thus, the link between early DD and next AP, missed in earlier studies, is ensured by a precisely physiologically regulated Ca2+ clock within pacemaker cells that integrates multiple Ca2+-dependent functions and rhythmically ignites APs during late DD via LCRs-I NCX coupling.