Intercellular delivery of therapeutic oligonucleotides.

One promising approach to cancer therapeutics is to induce changes in gene expression that either reduce cancer cell proliferation or induce cancer cell death. Therefore, delivering oligonucleotides (siRNA/miRNA) that target specific genes or gene programs might have a potential therapeutic benefit. The aim of this study was to examine the potential of cell-based delivery of oligonucleotides to cancer cells via two naturally occurring intercellular pathways: gap junctions and vesicular/exosomal traffic. We utilized human mesenchymal stem cells (hMSCs) as delivery cells and chose to deliver in vitro two synthetic oligonucleotides, AllStars HS Cell Death siRNA and miR-16 mimic, as toxic (therapeutic) oligonucleotides targeting three cancer cell lines: prostate (PC3), pancreatic (PANC1) and cervical (HeLa). Both oligonucleotides dramatically reduced cell proliferation and/or induced cell death when transfected directly into target cells and delivery hMSCs. The delivery and target cells we chose express gap junction connexin 43 (Cx43) endogenously (PC3, PANC1, hMSC) or via stable transfection (HeLaCx43). Co-culture of hMSCs (transfected with either toxic oligonucleotide) with any of Cx43 expressing cancer cells induced target cell death (~20% surviving) or senescence (~85% proliferation reduction) over 96 hours. We eliminated gap junction-mediated delivery by using connexin deficient HeLaWT cells or knocking out endogenous Cx43 in PANC1 and PC3 cells via CRISPR/Cas9. Subsequently, all Cx43 deficient target cells co-cultured with the same toxic oligonucleotide loaded hMSCs proliferated, albeit at significantly slower rates, with cell number increasing on average ~2.2-fold (30% of control cells) over 96 hours. Our results show that both gap junction and vesicular/exosomal intercellular delivery pathways from hMSCs to target cancer cells deliver oligonucleotides and function to either induce cell death or significantly reduce their proliferation. Thus, hMSC-based cellular delivery is an effective method of delivering synthetic oligonucleotides that can significantly reduce tumor cell growth and should be further investigated as a possible approach to cancer therapy.

Lens Connexin Channels Show Differential Permeability to Signaling Molecules.

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

Lens Connexin Channels Have Differential Permeability to the Second Messenger cAMP.

Purpose: Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation. Methods: We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule. Results: cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46. Conclusions: These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.

Intercellular communication via gap junction channels between chondrocytes and bone cells.

Cell-to-cell communication between bone, cartilage and the synovial membrane is not fully understood and it is only attributed to the diffusion of substances through the extracellular space or synovial fluid. In this study, we found for the first time that primary bone cells (BCs) including osteocytes, synovial cells (SCs) and chondrocytes (CHs) are able to establish cellular contacts and to couple through gap junction (GJ) channels with connexin43 (Cx43) being dominant. Transwell co-culture and identification by mass spectrometry revealed the exchange of essential amino acids, peptides and proteins including calnexin, calreticulin or CD44 antigen between contacting SCs, BCs and CHs. These results reveal that CHs, SCs and BCs are able to establish intercellular connections and to communicate through GJ channels, which provide a selective signalling route by the direct exchange of potent signalling molecules and metabolites.

Cyclic nucleotide permeability through unopposed connexin hemichannels.

Cyclic adenosine monophosphate (cAMP) is a well-known intracellular and intercellular second messenger. The membrane permeability of such molecules has potential importance for autocrine-like or paracrine-like delivery. Here experiments have been designed to demonstrate whether gap junction hemichannels, composed of connexins, are a possible entrance pathway for cyclic nucleotides into the interior of cells. HeLa cells stably expressing connexin43 (Cx43) and connexin26 (Cx26) were used to study the cyclic nucleotide permeability of gap junction hemichannels. For the detection of cAMP uptake, the cells were transfected using the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH) as the cAMP sensor. SpIH derived currents (I m) were recorded in whole-cell/perforated patch clamp configuration. Perfusion of the cells in an external K(+) aspartate(-) (KAsp) solution containing 500 µM cAMP and no extracellular Ca(2) (+), yielded a five to sevenfold increase in the I m current level. The SpIH current increase was associated with detectable hemichannel current activity. Depolarization of cells in Ca(2) (+)-free NaCl perfusate with 500 µM cAMP also induced a SpIH current increase. Elevating extracellular Ca(2) (+) to mM levels inhibited hemichannel activity. Perfusion with a depolarizing KAsp solution containing 500 µM cAMP and 2 mM Ca(2) (+) did not increase SpIH currents. The addition of the gap junction blocker carbenoxolone to the external solution inhibited cAMP uptake. Both cell depolarization and lowered extracellular Ca(2) (+) increase the open probability of non-junctional hemichannels. Accordingly, the SpIH current augmentation was induced by the uptake of extracellular cAMP via open membrane hemichannels in Cx43 and Cx26 expressing cells. The data presented here show that hemichannels of Cx43 and Cx26 are permeable to cAMP, and further the data suggest that hemichannels are, in fact, a potential pathway for cAMP mediated cell-to-cell signaling.

Can gap junctions deliver?

In vivo delivery of small interfering RNAs (siRNAs) to target cells via the extracellular space has been hampered by dilution effects and immune responses. Gap junction-mediated transfer between cells avoids the extracellular space and its associated limitations. Because of these advantages cell based delivery via gap junctions has emerged as a viable alternative for siRNA or miRNA delivery. Here we discuss the advantages and disadvantages of extracellular delivery and cell to cell delivery via gap junction channels composed of connexins. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome.

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.

Stimulating cardiac muscle by light: cardiac optogenetics by cell delivery.

BACKGROUND: After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. METHODS AND RESULTS: We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. CONCLUSIONS: Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

Regulation of gap junction coupling in bovine ciliary epithelium.

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 +/- 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R(f)) in recipient to donor cell was 0.47 +/- 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R(f) by approximately 60% to 0.20 +/- 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 microM) also reduced LY dye transfer by approximately 60%, reducing R(f) from 0.41 +/- 0.05 (n = 15) to 0.17 +/- 0.05 (n = 20) after 10 min. Junctional currents were lowered by approximately 50% (n = 6) after 10-min perfusion with 500 microM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 microM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

Connexin26 deafness associated mutations show altered permeability to large cationic molecules.

Intercellular communication is important for cochlear homeostasis because connexin26 (Cx26) mutations are the leading cause of hereditary deafness. Gap junctions formed by different connexins have unique selectivity to large molecules, so compensating for the loss of one isoform can be challenging in the case of disease causing mutations. We compared the properties of Cx26 mutants T8M and N206S with wild-type channels in transfected cells using dual whole cell voltage clamp and dye flux experiments. Wild-type and mutant channels demonstrated comparable ionic coupling, and their average unitary conductance was approximately 106 and approximately 60 pS in 120 mM K(+)-aspartate(-) and TEA(+)-aspartate(-) solution, respectively, documenting their equivalent permeability to K(+) and TEA(+). Comparison of cAMP, Lucifer Yellow (LY), and ethidium bromide (EtBr) transfer revealed differences in selectivity for larger anionic and cationic tracers. cAMP and LY permeability to wild-type and mutant channels was similar, whereas the transfer of EtBr through mutant channels was greatly reduced compared with wild-type junctions. Altered permeability of Cx26 to large cationic molecules suggests an essential role for biochemical coupling in cochlear homeostasis.

Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides.

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins.

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.

Nontransformed cells can normalize gap junctional communication with transformed cells.

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers.

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Normal cells control the growth of neighboring transformed cells independent of gap junctional communication and SRC activity.

The growth of many types of cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed cells suppress the growth of neighboring transformed cells. Direct physical contact between transformed and nontransformed cells was required for growth suppression of transformed cells in this system; communication by diffusible factors was not sufficient. However, significant gap junctional communication was not required, indicating that other intercellular junctions mediated this growth regulatory response. We also report that the Src kinase activity in transformed cells was not directly inhibited by contact with nontransformed cells. Instead, nontransformed cells increased the expression of serum deprivation-response protein and the transcription factor four and a half LIM domain 1 in tumor cells. In addition, these results suggest mechanisms by which normal cells may block Wnt signaling, inhibit insulin-like growth factor activity, and promote host recognition of neighboring tumor cells.

Gap junction uncoupling protects the heart against ischemia.

BACKGROUND: Many stimuli can successfully protect the heart against ischemia. We investigated whether gap junction uncoupling before ischemia was myoprotective. We also studied the function of the adenosine triphosphate-dependent potassium channel, which has been implicated in the mechanism of pharmacologic preconditioning, with respect to gap junction physiology. METHODS: Twenty-eight rabbit hearts were placed on a Langendorff perfusion apparatus. Five were given a 5-minute infusion of 1 mmol/L heptanol (a gap junction uncoupler), 5 were given 10 micromol/L 2,3-butanedione monoxime (an electromechanical uncoupler), and 6 were given no drug. The left anterior descending coronary artery was then occluded for 1 hour and reperfused for 2 hours. Six hearts received 10 micromol/L glybenclamide before heptanol to evaluate the role of the adenosine triphosphate-dependent potassium channel. Six hearts underwent ischemic preconditioning with 2 cycles of 5 minutes of global ischemia and reperfusion. Action-potential duration of the ischemic zone, left ventricular developed pressure, and coronary flow were measured continuously. Infarct size was determined at the end of reperfusion. RESULTS: Heptanol significantly reduced infarct size (from 46% +/- 2% to 22% +/- 5%, P <.01), an effect that was not prevented by glybenclamide. Butanedione monoxime decreased developed pressure but did not significantly reduce infarct size (46% +/- 5% vs 46% +/- 2%, P = not significant). There were no differences among groups with regard to developed pressure or action-potential duration. CONCLUSION: Directly blocking gap junctions preconditions the heart. This protection is not a direct result of a decrease in developed pressure before a prolonged ischemic period nor is it achieved through a mechanism involving the adenosine triphosphate-dependent potassium channel.