Adenoviral Gene Transfer Research Articles

S‐Palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of GC‐1 in Cardiomyocytes

IntroductionThe nitric oxide (NO) receptor soluble guanylyl cyclase (GC‐1) was first identified as a cytosolic enzyme but is now known to localize to various cell membrane microdomains. We reported that in normal hearts, GC‐1 localizes to caveolae, where it exhibits enhanced NO‐responsiveness. In failing hearts, caveolae‐localization of GC‐1 is disrupted and GC‐1 outside of caveolae is oxidized. How GC‐1 dynamically localizes to and away from caveolae is unknown. We hypothesized that S‐palmitoylation, a reversible, enzyme‐catalyzed, post‐translational modification that enhances membrane affinity of protein substrates, mediates caveolae‐localization and modulates redox regulation of GC‐1.MethodsTo examine S‐palmitoylation of GC‐1 in the failing heart, we subjected adult male C57BL/6 mice to transverse aortic constriction (TAC) vs thoracotomy (Sham) and performed modified acyl‐biotin exchange (ABE) assays. Expression of palmitoyl‐transferases ZDHHC1, ‐16, and ‐24 were also assessed in mouse hearts at the transcript and protein levels. Using computational bioinformatics, we identified 5 conserved cysteines of potential S‐palmitoylation in the GC‐1α subunit. We created cysteine‐serine mutants of GC‐1α at each and all of the 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). We expressed the mutants and WT GC‐1α in neonatal rat cardiomyocytes (NRCM) via adenoviral mediated gene transfer and compared their palmitoylation status by ABE. We also assessed caveolae‐localization of GC‐1α (CS* vs WT) by confocal microscopy and its oxidation by redox Western.ResultsIn TAC hearts, S‐palmitoylation of GC‐1α was diminished by 50% compared to Sham (p=0.02). Likewise, ZDHHC16 was also downregulated in TAC by 31% (p=0.01) at the transcript level and by 56% (p=0.03) at the protein level, relative to Sham. In NRCMs, S‐palmitoylation of CS* was profoundly diminished (6.9±2.0 % of WT, p<0.001), while that of C15S, C176S, and C497S were each modestly diminished (23.8±8.4%, p=0.03; 23.2±6.6%, p=0.01; 22.8±6.0%, p=0.01, respectively of WT). S‐palmitoylation of C3S and C595S were similar to WT. Confocal microscopy of NRCMs revealed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed a trend towards increased cysteine oxidation of CS* vs WT upon H2O2 treatment (1.5‐fold increase, p=0.05).ConclusionsOur data suggest that S‐palmitoylation of GC‐1α at C15, C176, and C497 mediates GC‐1 caveolae‐localization and may reciprocally regulate cysteine post‐translational modifications of GC‐1.Support or Funding Information5R01HL138528 to EJT.

Adenoviral Gene Transfer of Gremlin Modulates Vascular Endothelial Growth Factor-A-Induced Angiogenesis in Porcine Myocardium.

Coronary artery disease is a major cause of death and disability worldwide. New therapies are needed for patients who do not benefit or are not suitable for current treatments. Angiogenic gene therapy using vascular endothelial growth factors (VEGFs) has shown potential in preclinical trials. However, undesired side effects, such as increased permeability, limit their therapeutic potential. The aim of this study was to investigate if adenoviral gene transfer of a VEGF receptor 2 (VEGFR-2) ligand Gremlin, given simultaneously with VEGF-A, could modulate VEGFR-2-mediated increase in permeability without impairing the angiogenic effect of VEGF-A gene therapy. Gene transfers were done in pigs (n = 22) using endocardial injections with an endovascular injection catheter. Animals were divided in three groups receiving adenoviral (Ad) VEGF-A (n = 10), Gremlin (n = 6), or VEGF-A+Gremlin (n = 6) gene therapy. Animals were sacrificed and samples collected 6 days later for histological, safety, and permeability analyses. The mean capillary area was significantly increased in both treatment groups with AdVEGF-A when compared with the AdGremlin group. Also, the capillary area was significantly larger in AdVEGF-A group without AdGremlin. No significant differences in tissue permeability were observed using modified Miles assay between AdVEGF-A and AdVEGF-A+AdGremlin groups. However, cardiac tamponade and sudden cardiac deaths were observed only in the AdVEGF-A group. AdVEGF-A induces strong angiogenesis in porcine myocardium. Our results suggest that AdGremlin can limit the side effects of AdVEGF-A therapy, even though no direct effect on tissue permeability could be demonstrated. This could enable the use of larger AdVEGF-A doses to increase the treatment area and angiogenic effects without adverse side effects.

Involvement of Yes-associated protein 1 (YAP1) in doxorubicin-induced cytotoxicity in H9c2 cardiac cells.

Cardiac cell death is one of the major events implicated in doxorubicin-induced cardiotoxicity, which leads to heart failure. We recently reported that Yes-associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin-induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin-induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin-treated mice with increased caspase-3 activation. Doxorubicin also caused cell death by increasing caspase-3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase-3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1-5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin-induced cell death by decreasing caspase-3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin- and YAP1 short interfering RNA (siRNA)-treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin-induced cardiac cell death is mediated in part by down-regulation of YAP1 and YAP1-targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin-induced cardiotoxicity.

Adenoviral βARKct Cardiac Gene Transfer Ameliorates Postresuscitation Myocardial Injury in a Porcine Model of Cardiac Arrest.

The aim of the study was to determine whether the inhibition of the G-protein-coupled receptor kinase 2 by adenoviral βARKct cardiac gene transfer can ameliorate postresuscitation myocardial injury in pigs with cardiac arrest (CA) and explore the mechanism of myocardial protection. Male landrace domestic pigs were randomized into the sham group (anesthetized and instrumented, but ventricular fibrillation was not induced) (n = 4), control group (ventricular fibrillation 8 min, n = 8), and βARKct group (ventricular fibrillation 8 min, n = 8). Hemodynamic parameters were monitored continuously. Blood samples were collected at baseline, 30 min, 2 h, 4 h, and 6 h after the return of spontaneous circulation (ROSC). Left ventricular ejection fraction was assessed by echocardiography at baseline and 6 h after ROSC. These animals were euthanized, and the cardiac tissue was removed for analysis at 6 h after ROSC. Compared with those in the sham group, left ventricular +dp/dtmax, -dp/dtmax, cardiac output (CO), and ejection fraction (EF) in the control group and the βARKct group were significantly decreased at 6 h after the restoration of spontaneous circulation. However, the βARKct treatment produced better left ventricular +dp/dtmax, -dp/dtmax, CO, and EF after ROSC. The βARKct treatment also produced lower serum cardiac troponin I, CK-MB, and lactate after ROSC. Furthermore, the adenoviral βARKct gene transfer significantly increased β1 adrenergic receptors, SERCA2a, RyR2 levels, and decreased GRK2 levels compared to control. The inhibition of GRK2 by adenoviral βARKct cardiac gene transfer can ameliorate postresuscitation myocardial injury through beneficial effects on restoring the sarcoplasmic reticulum Ca-handling proteins expression and upregulating the β1-adrenergic receptor level after cardiac arrest.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

Adenoviral gene transfer of a single-chain IL-23 induces psoriatic arthritis-like symptoms in NOD mice.

Previously, we demonstrated that intratumoral delivery of adenoviral vector encoding single-chain (sc)IL-23 (Ad.scIL-23) was able to induce systemic antitumor immunity. Here, we examined the role of IL-23 in diabetes in nonobese diabetic mice. Intravenous delivery of Ad.scIL-23 did not accelerate the onset of hyperglycemia but instead resulted in the development of psoriatic arthritis. Ad.scIL-23-treated mice developed erythema, scales, and thickening of the skin, as well as intervertebral disc degeneration and extensive synovial hypertrophy and loss of articular cartilage in the knees. Immunological analysis revealed activation of conventional T helper type 17 cells and IL-17-producing γδ T cells along with a significant depletion and suppression of T cells in the pancreatic lymph nodes. Furthermore, treatment with anti-IL-17 antibody reduced joint and skin psoriatic arthritis pathologies. Thus, these Ad.scIL-23-treated mice represent a physiologically relevant model of psoriatic arthritis for understanding disease progression and for testing therapeutic approaches.-Flores, R. R., Carbo, L., Kim, E., Van Meter, M., De Padilla, C. M. L., Zhao, J., Colangelo, D., Yousefzadeh, M. J., Angelini, L. A., Zhang, L., Pola, E., Vo, N., Evans, C. H., Gambotto, A., Niedernhofer, L. J., Robbins, P. D. Adenoviral gene transfer of a single-chain IL-23 induces psoriatic arthritis-like symptoms in NOD mice.

Canine adenoviral vector-mediated gene transfer to the guinea pig brain

Pre-clinical testing of gene transfer tools is often first conducted in small animal models such as laboratory mice or rats, before extension into a larger animal such as non-human primates. The guinea pig brain may provide an intermediary model in which to examine neurodevelopmental processes given that guinea pigs are considered precocial. Here, we characterized the efficacy and biodistribution following injection of a canine adenovirus type 2 (CAV-2) vector in the young adult guinea pig brain. Despite the development of mild fevers post-treatment in the vector-treated animals, widespread transduction was evident throughout the brain parenchyma. Neutralizing antibodies against CAV-2 were not detected. Our data demonstrate that the combination of CAV-2 vectors and guinea pigs provide a clinically relevant model for evaluation of therapeutics.

The FMS-like tyrosine kinase-3 ligand/lung dendritic cell axis contributes to regulation of pulmonary fibrosis

RationaleDendritic cells (DC) accumulate in the lungs of patients with idiopathic lung fibrosis, but their pathogenetic relevance is poorly defined.ObjectivesTo assess the role of the FMS-like tyrosine kinase-3 ligand (Flt3L)-lung...

Interplay between the Yes‐Associated protein and the matricellular protein CCN1 Regulates the phenotypical plasticity of endothelial cells in developing blood vessels

CCN1 is a heparin and integrin‐binding matricellular protein which localizes pericellularly and acts primarily on cells that produce it. CCN1 is highly expressed at sites of active angiogenesis and tissue repair. Our studies in conditional knockout mouse models, demonstrate that endothelium‐specific deletion of the CCN1 gene results in severe vascular defects, most notably uncontrolled angiogenic sprouting and increased endothelial cell (EC) proliferation, resulting in the formation of a denser, widely lumenized vascular network. In this study, we investigated the regulatory mechanisms and signaling pathways involved in CCN1 gene expression and activity during angiogenesis. We found that the CCN1 gene is a target of the transcriptional coactivator Yes‐Associated protein (YAP), which acts in concert with other co‐activators such as myocardin‐related transcription factor (MRTF)‐A to activate the CCN1 promoter in angiogenic ECs. Loss of YAP function in mice is associated with reduced CCN1 promoter enrichment with YAP and decreased CCN1 gene expression. YAP is a key checkpoint of the Hippo pathway controlling cell growth, differentiation and cell‐cell junctions. It comprises a kinase cascade that inactivates by phosphorylation YAP leading to its cytoplasmic sequestration and growth inhibition. Loss of function of CCN1 in mice was associated with accumulation of active nonphosphorylated YAP in developing blood vessels. Consequently, growing ECs incorporate into preformed vessels and are no longer directed toward angiogenic sprouts, resulting in the formation of a thicker denser vasculature. In cultured ECs, a sustained expression of CCN1 through adenoviral gene transfer increases YAP phosphorylation and reduced its localization into the nucleus. CCN1‐integrin signaling through focal adhesion and the actin cytoskeleton provides the cells with a soft compliant matrix putting them in in low contractility, YAP repressive cytoskeletal states. This crosstalk between CCN1 and YAP regulates phenotypical plasticity of ECs and promotes correct sprouting and branching patterns of developing blood vessels.Support or Funding InformationR01EY022091‐05A1R01EY024998‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts.

Key points Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.PKA phosphorylation attenuates I SK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i.This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked I SK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of I SK via reduction Ca2+/voltage‐dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465.

Apolipoprotein M suppresses the phenotypes of IgA nephropathy in hyper-IgA mice.

Because the association between sphingosine 1-phosphate (S1P)/apolipoprotein M (ApoM) and chronic kidney diseases has not been established, we investigated the involvement of S1P/ApoM in the phenotypes of IgA nephropathy in hyper-IgA (HIGA) mice. The overexpression of ApoM in adenoviral gene transfer ameliorated the phenotypes of IgA nephropathy in HIGA mice, whereas the knockdown of ApoM with siRNA caused deterioration. When ApoM-overexpressing HIGA mice were treated with VPC23019, an antagonist against S1P receptor 1 (S1P1) and 3 (S1P3), we observed that the protective effects of ApoM were reversed, whereas JTE013, an antagonist against S1P2, did not inhibit the effects. We also found that S1P bound to albumin accelerated the proliferation of MES13 cells and the fibrotic changes of HK2 cells, which were inhibited by JTE013, whereas S1P bound to ApoM suppressed these changes, which were inhibited by VPC23019. These results suggest that S1P bound to ApoM possesses properties protective against the phenotypes of IgA nephropathy through S1P1 and S1P3, whereas S1P bound to albumin exerts deteriorating effects through S1P2. ApoM may be useful as a therapeutic target to treat or retard the progression of IgA nephropathy.-Kurano, M., Tsuneyama, K., Morimoto, Y., Nishikawa, M., Yatomi, Y. Apolipoprotein M suppresses the phenotypes of IgA nephropathy in hyper-IgA mice.

DPP10 is a new regulator of Nav1.5 channels in human heart

BackgroundCardiac accessory β-subunits are part of macromolecular Nav1.5 channel complexes modulating biophysical properties and contributing to arrhythmias. Recent studies demonstrated the structural interaction between β-subunits of Na+ (Nav1.5) and K+ (Kv4.3) channels. Here, we identified the dipeptidyl peptidase-like protein-10 (DPP10), which is known to modulate Kv4.3-current kinetics, as a new regulator of Nav1.5 channels. MethodsWe assessed DPP10 expression in the healthy and diseased human heart and we studied the functional effects of DPP10 on the Na+ current in isolated rat cardiomyocytes expressing DPP10 after adenoviral gene-transfer (DPP10ad). ResultsDPP10 mRNA and proteins were detected in human ventricle, with higher levels in patients with heart failure. In rat cardiomyocytes, DPP10ad significantly reduced upstroke velocity of action potentials indicating reduction in Na+-current density. DPP10 significantly shifted the voltage-dependent Na+ channel activation and inactivation curve to more positive potentials, resulting in greater availability of Na+ channels for activation, along with increasing window Na+ current. In addition, time-to-peak Na+ current was reduced, whereas time course of recovery from inactivation was significantly accelerated by DPP10ad. DPP10 co-immunoprecipitated with Nav1.5 channels in human ventricles, confirming their physical interaction. ConclusionWe provide first evidence that DPP10 interacts with Nav1.5 channels, linking Na+- and K+-channel complexes in the heart. Our data suggest that increased ventricular DPP10 expression in heart failure might promote arrhythmias by decreasing peak Na+ current, while increasing window Na+ current and channel re-openings due to accelerated recovery from inactivation.

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option.Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection.Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers.Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

Prevention of Corneal Neovascularization by Adenovirus Encoding Human Vascular Endothelial Growth Factor Soluble Receptor (s-VEGFR1) in Lacrimal Gland.

The aims of this study were (1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (s-VEGFR1) gene transfer to the lacrimal gland (LG); (2) to investigate whether expression of s-VEGFR1 prevents corneal neovascularization (CNV) induced by alkali burns; and (3) to evaluate the safety of the procedure. AdVEGFR1 vectors (25 μL, 1 × 1010 pfu/mL) were injected in the right LGs of rats and were compared with AdNull vector (25 μL, 1 × 1010 pfu/mL) or 25 μL of saline (Control) before cornea alkali burns with 1 M NaOH. After 7 days, CNV was documented at the slit lamp. Tear secretion was measured with phenol red threads. The animals were tested for s-VEGFR1 mRNA and protein in the LG by quantitative (q)PCR and immunohistochemistry staining, respectively. qPCR was used to compare the mRNA levels of IL-1β, IL-6, and TNF-α in the LG and ipsilateral trigeminal ganglion (TG). Ad-VEGFR1 transfected 83% (10/12) of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals in the Ad-VEGFR1 group, compared with the Ad-Null (3:10) and Control groups (1:10) (P = 0.0317). The tear secretion and cytokine mRNA levels in the LG and TG were similar in all three groups (P > 0.05). Adenoviral vector gene transfer was safe for LG structure and function. The LG as the target tissue showed local expression of human s-VEGFR1, and CNV was prevented in most of the eyes exposed to alkali burns.

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting.

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105+ fibroblast cells through the Smad and MAPK pathways.

The skin, as the largest organ of the human body, is an important source of stromal stem cells with multipotent differentiation potential. CD105+ mesenchymal stem cells exhibit a higher level of stemness than CD105− cells. In the present study, human dermal-derived CD105+ fibroblast cells (CD105+ hDDFCs) were isolated from human foreskin specimens using immunomagnetic isolation methods to examine the role of bone morphogenetic protein (BMP)-7 in osteogenic differentiation. Adenovirus-mediated recombinant BMP7 expression enhanced osteogenesis-associated gene expression, calcium deposition, and alkaline phosphatase activity. Investigation of the underlying mechanisms showed that BMP7 activated small mothers against decapentaplegic (Smad) and p38/mitogen-activated protein kinase signaling in CD105+ hDDFCs. The small interfering RNA-mediated knockdown of Smad4 or inhibition of p38 attenuated the BMP7-induced enhancement of osteogenic differentiation. In an in vivo ectopic bone formation model, the adenovirus-mediated overexpression of BMP7 enhanced bone formation from CD105+ hDDFCs. Taken together, these data indicated that adenoviral BMP7 gene transfer in CD105+ hDDFCs may be developed as an effective tool for bone tissue engineering.

Human Vascular Endothelial Growth Factor A165 Expression Induces the Mouse Model of Neovascular Age-Related Macular Degeneration.

Vascular endothelial growth factor (VEGF) expression induces age-related macular degeneration (AMD), which is a common vision-threatening disease due to choroidal neovascularization and a fibrovascular membrane. We describe a mouse model of neovascular AMD with the local expression of human VEGF-A165 in the eye. We use a transgenic mouse in which human VEGF-A165 has been silenced with the loxP-STOP fragment. The choroidal neovascularization and human VEGF-A165 expression in the mouse are induced by subretinal adenoviral Cre gene delivery. Cre gene transfer is compared with adenoviral LacZ gene transfer control. We characterize the AMD phenotype and changes in the vasculature by using fluorescein angiography, optical coherence tomography, and immunohistochemistry. At early time points, mice exhibit increases in retinal thickness (348 ± 114 µm vs. 231 ± 32 µm) and choroidal neovascularization area (12000 ± 15174 µm2 vs. 2169 ± 3495 µm2) compared with the control. At later time points, choroidal neovascularization develops into subretinal fibrovascular membrane. Human VEGF-A165 expression lasts several weeks. In conclusion, the retinas display vascular abnormalities consistent with choroidal neovascularization. Together with immunohistochemical findings, these changes resemble clinical AMD-like ocular pathologies. We conclude that this mouse model of Cre-induced choroidal neovascularization is useful for mimicking the pathogenesis of AMD, studying the effects of human VEGF-A165 in the retina, and evaluating anti-VEGF treatments for choroidal neovascularization.

Knockdown of CLC-3 in the hippocampal CA1 impairs contextual fear memory

Previous studies support a critical role of hippocampus in contextual fear memory. Structural and functional alterations of hippocampus occur frequently in posttraumatic stress disorders (PTSD). Recent reports reveal that knockout of CLC-3, a member of the CLC family of anion channels and transporters, leads to neuronal degeneration and loss of hippocampus. However, the role of CLC-3 in contextual fear memory remains unknown. Using adenovirus and adeno-associated virus gene transfer to knockdown CLC-3 in hippocampal CA1, we investigate the role of CLC-3 in contextual fear memory. CLC-3 expression is increased in hippocampal CA1 after formation of long-term contextual fear memory. Knockdown of CLC-3 by adenovirus infusion in hippocampal CA1 significantly attenuates the contextual fear memory, reduces spine density, induces defects of excitatory synaptic ultrastructure showed by the decreased PSD length, PSD thickness and active zone length, and impairs L-LTP induction and maintenance. Knockdown of CLC-3 also induces the synaptic NMDAR subunit composition to an increased GluN2A/GluN2B ratio pattern and reduces the activity of CaMKII-α. Furthermore, selectively knockdown of CLC-3 in excitatory neurons by adeno-associated virus driven from CaMKII-α promoter is sufficient to impair long-term contextual fear memory. These findings highlight that CLC-3 in hippocampal CA1 is necessary for contextual fear memory.

Regulation of the metabolism of apolipoprotein M and sphingosine 1-phosphate by hepatic PPARγ activity.

Apolipoprotein M (apoM) is a carrier and a modulator of sphingosine 1-phosphate (S1P), an important multifunctional bioactive lipid. Since peroxisome proliferator-activated receptor γ (PPARγ) is reportedly associated with the function and metabolism of S1P, we investigated the modulation of apoM/S1P homeostasis by PPARγ. First, we investigated the modulation of apoM and S1P homeostasis by the overexpression or knockdown of PPARγ in HepG2 cells and found that both the overexpression and the knockdown of PPARγ decreased apoM expression and S1P synthesis. When we activated or suppressed the PPARγ more mildly with pioglitazone or GW9662, we found that pioglitazone suppressed apoM expression and S1P synthesis, while GW9662 increased them. Next, we overexpressed PPARγ in mouse liver through adenoviral gene transfer and observed that both the plasma and hepatic apoM levels and the plasma S1P levels decreased, while the hepatic S1P levels increased, in the presence of enhanced sphingosine kinase activity. Treatment with pioglitazone decreased both the plasma and hepatic apoM and S1P levels only in diet-induced obese mice. Moreover, the overexpression of apoM increased, while the knockdown of apoM suppressed PPARγ activities in HepG2 cells. These results suggested that PPARγ regulates the S1P levels by modulating apoM in a bell-shaped manner, with the greatest levels of apoM/S1P observed when PPARγ was mildly expressed and that hepatic apoM/PPARγ axis might maintain the homeostasis of S1P metabolism.

SOCS-1 Suppresses Inflammation Through Inhibition of NALP3 Inflammasome Formation in Smoke Inhalation-Induced Acute Lung Injury.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality rates. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of proinflammatory cytokine signaling. We have found that adenoviral gene transfer of SOCS-1 ameliorates smoke inhalation-induced lung injury in C57BL/6 mice. We also found that the release of adenosine triphosphate (ATP) was increased post smoke exposure, while oxidized ATP, an inhibitor of purinergic P2X7 receptor, suppressed smoke-induced NALP3 inflammasome assembly, caspase-1 activation, and K+ efflux. Similar to oxidized ATP, high protein level of SOCS-1 dampened the formation of NALP3 inflammasome and the activation of caspase-1 and IL-1β induced by smoke exposure in mouse alveolar macrophages. In conclusion, SOCS-1 relieves smoke inhalation-induced pulmonary inflammation and injury by inhibiting NALP3 inflammasome formation.