Light-Triggered Drug Release from Red Blood Cells Suppresses Arthritic Inflammation.

Arthritis is a leading cause of disability in adults, which can be intensely incapacitating. The location and intensity of the pain is both subjective and challenging to manage. Consequently, patient-directed delivery of anti-inflammatories is an essential component of future therapeutic strategies for the management of this disorder. We describe the design and application of a light responsive red blood cell (RBC) conveyed dexamethasone (Dex) construct that enables targeted drug delivery upon illumination of the inflamed site. The red wavelength (650 nm) responsive nature of the phototherapeutic was validated using tissue phantoms mimicking the light absorbing properties of various skin types. Furthermore, photoreleased Dex has the same impact on cellular responses as conventional Dex. Murine RBCs containing the photoactivatable therapeutic display comparable circulation properties as fluorescently labelled RBCs. In addition, a single dose of light-targeted Dex delivery is 5-fold more effective in suppressing inflammation than the parent drug, delivered serially over multiple days. These results are consistent with the notion that the circulatory system be used as an on-command drug depot, providing the means to therapeutically target diseased sites both efficiently and effectively.

Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

Optogenetic perturbation of the biochemical pathways that control cell behavior.

Optogenetic tools provide a level of spatial and temporal resolution needed to shed new light on dynamic intercellular processes. In this chapter we outline specific protocols for applying these tools to cell motility (optogenetic cofilin), apoptosis [optogenetic Bcl-like protein 4 (Bax)], and protein kinase-mediated signaling pathways [optogenetic cAMP-dependent protein kinase (PKA)]. The activity of these optogenetic species is regulated by the light-mediated dimerization of a cryptochrome/Cib protein pair, which controls the intracellular positioning of the protein of interest. The light induced recruitment of cofilin to the cytoskeleton is utilized for directed migration studies and filopodial dynamics. Light-triggered migration of Bax to the outer mitochondrial membrane induces cellular collapse and eventual apoptosis. Finally, the light-mediated movement of PKA to specific intracellular compartments offers the means to assess the consequences of PKA activity in a site-specific fashion via phosphoproteomic analysis.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Dasatinib Is Preferentially Active in the Activated B-Cell Subtype of Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and at least one-third of its patients relapse after treatment with the current chemotherapy regimen, R-CHOP. By gene-expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL. Patients with the ABC subtype have a worse prognosis than those with GCB, and the subtype is defined by chronic, over-active signaling through the B-cell receptor and NF-κB pathways. We examined the effects of the Src family kinase (SFK) inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines and found that the former are much more sensitive to dasatinib than the latter. However, using multiplexed inhibitor bead coupled to mass spectrometry (MIB/MS) kinome profiling and Western blot analysis, we found that both subtypes display inhibition of the SFKs in response to dasatinib after both short- and long-term treatment. The MIB/MS analyses revealed that several cell-cycle kinases, including CDK4, CDK6, and the Aurora kinases, are down-regulated by dasatinib treatment in the ABC, but not in the GCB, subtype. The present findings have potential implications for the clinical use of dasatinib for the treatment of ABC DLBCL, either alone or in combination with other agents.

Cardioprotection via the skin: nociceptor-induced conditioning against cardiac MI in the NIC of time.

Timely reperfusion is still the most effective approach to limit infarct size in humans. Yet, despite advances in care and reduction in door-to-balloon times, nearly 25% of patients develop heart failure postmyocardial infarction, with its attendant morbidity and mortality. We previously showed that cardioprotection results from a skin incision through the umbilicus in a murine model of myocardial infarction. In the present study, we show that an electrical stimulus or topical capsaicin applied to the skin in the same region induces significantly reduced infarct size in a murine model. We define this class of phenomena as nociceptor-induced conditioning (NIC) based on the peripheral nerve mechanism of initiation. We show that NIC is effective both as a preconditioning and postconditioning remote stimulus, reducing infarct size by 86% and 80%, respectively. NIC is induced via activation of skin C-fiber nerves. Interestingly, the skin region that activates NIC is limited to the anterior of the T9-T10 vertebral region of the abdomen. Cardioprotection after NIC requires the integrity of the spinal cord from the region of stimulation to the thoracic vertebral region of the origin of the cardiac nerves but does not require that the cord be intact in the cervical region. Thus, we show that NIC is a reflex and not a central nervous system-mediated effect. The mechanism involves bradykinin 2 receptor activity and activation of PKC, specifically, PKC-α. The similarity of the neuroanatomy and conservation of the effectors of cardioprotection supports that NIC may be translatable to humans as a nontraumatic and practical adjunct therapy against ischemic disease. NEW & NOTEWORTHY This study shows that an electrical stimulus to skin sensory nerves elicits a very powerful cardioprotection against myocardial infarction. This stimulus works by a neurogenic mechanism similar to that previously elucidated for remote cardioprotection of trauma. Nociceptor-induced conditioning is equally potent when applied before ischemia or at reperfusion and has great potential clinically.

Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells.

Though experimental, stem cell transplantation has the potential to improve the condition of the heart after myocardial infarction. It does so by reducing infarct size and inducing repair of heart muscle and its blood supply. Mesenchymal stem cells (MSC) have been found to be effective in pre-clinical animal models and clinical trials, but the mechanisms by which they induce cardioprotection and repair are still not fully understood. Small extracellular vesicles known as exosomes are now recognized to be key mediators of beneficial MSC paracrine effects, and the concept that they transfer miRNA to change gene expression in recipient cells is of current therapeutic interest. We present complete deep miRNA sequencing of MSC exosome cargo, and found that of several cardioprotective miRNAs, miR-21a-5p was the most abundant. Because miR-21a-5p is a well-known cardioprotective miRNA, we investigated the hypothesis that MSC exosomes can cardioprotect the heart by increasing the level of miR-21a-5p in recipient cardiac cells, thereby downregulating expression of the pro-apoptotic gene products PDCD4, PTEN, Peli1 and FasL in the myocardium. Using miR-21 mimic transfection and treatment with wild type and miR-21a knockout MSC exosomes, we confirmed that exosomal miR-21a-5p is transferred into myocardium and is a major cardioprotective paracrine factor produced by MSCs acting via synergistic activity on multiple pathways. The data supports that residual cardioprotective effect may be due to other ncRNA or protein cargo. In silico analyses support that MSC exosomes may also contribute to angiogenesis, cell proliferation and other aspects of cardiac repair.

Activation of hERG3 channel stimulates autophagy and promotes cellular senescence in melanoma.

Ion channels play a major factor in maintaining cellular homeostasis but very little is known about the role of these proteins in cancer biology. In this work we have discovered that, the Kv11.3 (hERG3) a plasma-membrane potassium channel plays a critical role in the regulation of autophagy in a cancer cell model. We have found that pharmacologic stimulation of the Kv11.3 channel with a small molecule activator, NS1643 induced autophagy via activation of an AMPK-dependent signaling pathway in melanoma cell line. In addition, we have found that NS1643 produced a strong inhibition of cell proliferation by activating a cellular senescence program. Furthermore, inhibition of autophagy via siRNA targeting AMPK or treatment with hydroxychloroquine an autophagy inhibitor activates apoptosis in NS1643-treated cells. Thus, we propose that, Kv11.3 is a novel mediator of autophagy, autophagy can be a survival mechanism contributing to cellular senescence, and that use of a combinatorial pharmacologic approach of Kv11.3 activator with inhibitors of autophagy represents a novel therapeutic approach against melanoma.

Acute consumption of a high-fat diet prior to ischemia-reperfusion results in cardioprotection through NF-κB-dependent regulation of autophagic pathways.

Previous studies have demonstrated improvement of cardiac function occurs with acute consumption of a high-fat diet (HFD) after myocardial infarction (MI). However, no data exist addressing the effects of acute HFD upon the extent of injury after MI. This study investigates the hypothesis that short-term HFD, prior to infarction, protects the heart against ischemia-reperfusion (I/R) injury through NF-κB-dependent regulation of cell death pathways in the heart. Data show that an acute HFD initiates cardioprotection against MI (>50% reduction in infarct size normalized to risk region) after 24 h to 2 wk of HFD, but protection is completely absent after 6 wk of HFD, when mice are reported to develop pathophysiology related to the diet. Furthermore, cardioprotection after 24 h of HFD persists after an additional 24 h of normal chow feeding and was found to be dependent upon NF-κB activation in cardiomyocytes. This study also indicates that short-term HFD activates autophagic processes (beclin-1, LC-3) preischemia, as seen in other protective stimuli. Increases in beclin-1 and LC-3 were found to be NF-κB-dependent, and administration of chloroquine, an inhibitor of autophagy, abrogated cardioprotection. Our results support that acute high-fat feeding mediates cardioprotection against I/R injury associated with a NF-κB-dependent increase in autophagy and reduced apoptosis, as has been found for ischemic preconditioning.

Azelastine desensitization of transient receptor potential vanilloid 1: a potential mechanism explaining its therapeutic effect in nonallergic rhinitis.

BACKGROUND: Capsaicin, a prototypic transient receptor potential vanilloid 1 (TRPV1) agonist, has been shown to be more clinically effective in the treatment of nonallergic rhinitis (NAR) compared with other rhinitis subtypes. Azelastine has also been found to be clinically effective in the treatment of NAR but its mechanism(s) of action is still poorly elucidated. This study was designed to determine, using in vitro cell lines, whether topical therapies including azelastine have activity on TRPV1 ion channels similar to capsaicin. METHODS: The effects of capsaicin (1 µM), azelastine (30 µM), bepotastine (10 µM), olopatadine (10 µM), and fluticasone (200 µM) on TRPV1 channels using mice neuronal cells (Cath.a), as surrogates for submucosal sensory neurons, and human nasal epithelial cells (hNEC) were determined and compared. For azelastine, bepotastine, and capsaicin, which elicited an agonist effect on TRPV1, live cell [Ca(2+)] signaling in Cath.a cells and hNECs expressing TRPV1 were performed in the absence and presence of capsazepine at 10 µM (a TRPV1 antagonist) or using wild-type mouse embryonic fibroblasts (wtMEFs) that express TRPV1 ion channels and TRPV1 homozygous null mutant (TRPV1-/-) knockout MEF cells as controls to establish TRPV1 channel selectivity. As azelastine has previously been found clinically effective in NAR, additional experiments were performed to determine its ability to desensitize TRPV1 ion channels and its effect on regulating intracellular calcium homeostasis. RESULTS: Cath.a cells treated with azelastine, bepotastine, or capsaicin showed a significant increase in TRPV1-dependant (Ca(2+)) specific cytosolic fluorescence. Continuous treatment with azelastine or capsaicin resulted in desensitization of TRPV1 channels. In hNECs, azelastine stimulation resulted in Ca(2+) shifts from the cytosol to mitochondria and overexpression of hematopoietic cell-specific Lyn substrate 1-associated protein X1, which may thus be effective in cytosolic Ca(2+) homeostasis. CONCLUSION: Azelastine, similar to capsaicin, exhibits direct activity on TRPV1 ion channels that may represent a novel mechanistic pathway explaining its clinical efficacy in NAR.

NF-κΒ inhibition is ineffective in blocking cytokine-induced IL-8 production but P38 and STAT1 inhibitors are effective.

OBJECTIVE: In vitro but not in vivo evidence indicates that blockade of NF-κB is effective in reducing inflammation and production of IL-8. We hypothesized that the failure of in vitro experiments to predict in vivo outcome was due to the use of short time periods of observation and the use of single cytokines to stimulate NF-κB. METHODS: HEK cells with a NF-κB reporter gene or CaCo-2 cells were stimulated with CM (IL-1-ß; TNF-α, and IFN-γ) or individual cytokines in the presence and absence of NF-κB inhibitors, a STAT1 inhibitor, and/or a p38 MAPK inhibitor for periods up to 24 h. NF-κB activation, IL-8 production, and nitric oxide production were measured. RESULTS: CM-induced IL-8 production in HEK cells was additive to synergistic. CM enhanced production of IL-8 at 24 h but not 4 h was independent of NF-κB. The p38 inhibitor SB203580 and the STAT1 inhibitor EGCG blocked CM-induced IL-8 production at both early and late time periods. The NF-κB inhibitors PDTC and BAY11-7082 were found to increase CM-stimulated IL-8 production in Caco-2 cells at 24 h. CONCLUSIONS: Our data suggest an effective strategy to reduce IL-8 production is to block p38 or STAT1 rather than NF-κB.

Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation.

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and ß-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the ß-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+).

Coordinated post-transcriptional regulation of Hsp70.3 gene expression by microRNA and alternative polyadenylation.

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

Neutrophils, not monocyte/macrophages, are the major splenic source of postburn IL-10.

Burn induces myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature polymorphonuclear neutrophils (PMNs) and monocytes, which protect against infection. Previous work from our laboratory demonstrated that inflammatory monocytes (iMos) were the major MDSC source of TNF-α in the postburn spleen, and we hypothesized that they were also the major source of postburn IL-10. To test this hypothesis, we examined cytokine production by postburn CCR2 knockout (KO) mice, which have fewer iMos than burn wild-type (WT) splenocytes, but equal numbers of PMNs and F4/80 macrophages. Using cell sorting and/or intracellular cytokine techniques, we examined IL-10 production by postburn PMNs and iMos. Finally, we compared IL-10 production by postburn PMNs and iMos with culture-derived MDSCs. Splenocytes from postburn CCR2 KO mice produced less IL-6 and TNF-α than WT burn splenocytes in response to LPS, but KO and WT burn splenocytes produced equal amounts of IL-10 in response to peptidoglycan. Depletion of PMNs from postburn splenocytes led to reductions in IL-10 and increases in IL-6 and TNF-α in response to peptidoglycan, but not in response to LPS. Sorting or intracellular cytokine techniques gave consistent results: Burn PMNs made more IL-10 than sham PMNs and also more IL-10 than burn or sham iMos. Polymorphonuclear neutrophil and iMos subpopulations from culture-derived MDSCs produced the same cytokine profiles in response to LPS and peptidoglycan as did the PMNs and iMos from postburn spleens: PMNs made IL-10, whereas iMos made IL-6. Finally, LPS-induced mortality of burn mice was made worse by anti-Gr-1 depletion of all PMNs and 66% of iMos from burn mice. This suggests that PMNs play a primarily anti-inflammatory role in vitro and in vivo.

A ribonucleotide reductase inhibitor reverses burn-induced inflammatory defects.

Immature myeloid cells have been implicated as a source of postburn inflammation, and the appearance of these cells correlates with enhanced upregulation of hematopoiesis. The role of proliferative cells in postburn immune changes has not been directly tested. Gemcitabine, a ribonucleotide reductase inhibitor, has been shown to deplete proliferative immature myeloid cells in tumor models while sparing mature cells, leading to restored lymphocyte function and tumor regression. We treated burn mice at postburn day 6 (PBD6) with 120 mg/kg gemcitabine. On PBD8, splenocytes were taken and stimulated with LPS, peptidoglycan, or concanavalin A. The blood and spleen cell populations were enumerated by flow cytometry or automated cell counter. In addition, mice treated with gemcitabine were given LPS or infected with Pseudomonas aeruginosa at PBD8, and mortality was monitored. Gemcitabine depleted burn-induced polymorphonuclear leukocytes and inflammatory monocytes without affecting mature F4/80 macrophages. This was accompanied by reduced TNFα, IL-6, and IL-10 production by burn splenocytes. Burn splenocytes stimulated with mitogens exhibited increased nitric oxide production relative to sham mice. In vivo treatment of burn mice with gemcitabine blocked these burn-induced changes without damaging lymphocyte function. Treatment of burn mice with gemcitabine ameliorated burn-induced susceptibility to LPS and infiltration of polymorphonuclear leukocytes into the liver and lung. Finally, gemcitabine treatment blocked the protective effect of burn injury upon P. aeruginosa infection. Our report shows that proliferative cells are major drivers of postburn immune changes and provides evidence that implicates immature myeloid cells in these processes.