Abstract P224: β-arrestin2 Stimulates SERCA2a SUMOylation and Activity in Cardiac Myocytes to Promote Contractility

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Katie A McCrink ◽  
Jennifer Maning ◽  
Ava Brill ◽  
Angela Vu ◽  
Walter J Koch ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cardiac Contractility ◽  
Adapter Proteins ◽  
Inotropic Effects ◽  
Signal Transducers ◽  
Adenoviral Gene Transfer ◽  
Plasmic Reticulum ◽  
New Treatments

Background: Heart failure (HF) is the most lethal disease worldwide and new treatments are needed. The β 1 -adrenergic receptor (AR) mediates the positive inotropy of catecholamines, partly via Sarco(Endo)plasmic Reticulum Ca 2+ -ATPase (SERCA)-2a activation. Agonist-activated β 1 ARs, however, are desensitized/downregulated in human HF due to the actions of the βarrestins (βarrestin1 and 2). βarrestins are GPCR adapter proteins and signal transducers. βarrestin1 is by far the predominant isoform in the heart and reduces contractility by desensitizing the β 1 AR, whereas βarrestin2 is expressed at negligible levels and is beneficial post-myocardial infarction (MI), as it combats inflammation and apoptosis. Herein, we sought to investigate whether cardiac βarrestin2 exerts any inotropic effects. Methods: We used βarrestin knockout (KO) mouse hearts and also performed intra-cardiac adenoviral gene transfer of βarrestin2 (Adβarrestin2) in post-MI mice in vivo. For mechanistic signaling studies we used the cardiomyocyte cell line H9c2. Results: SERCA2a SUMO (small ubiquitin-like modifier)ylation and activity and, consequently, cardiac contractility were increased in βarrestin1 KO`s vs. WT`s post-MI. The opposite was true for βarrestin2 KO`s post-MI. Additionally, βarrestin2, but not βarrestin1, was found to directly bind SERCA2a and induce its SUMOylation and activation in mouse hearts in vivo, as well as in cardiomyocytes in vitro acutely in response to β 1 AR stimulation. Interestingly, βarrestin2 did not affect the classic β 1 AR cAMP-dependent pro-contractile signaling pathway in cardiomyocytes, again contrary to βarrestin1. Importantly, and consistent with these findings, Adβarrestin2 gene transfer in post-MI mouse hearts in vivo resulted in enhanced cardiac function (post-MI ejection fraction of Adβarrestin2 vs. control (AdGFP) mice: 40.3 + 1.3% vs. 23.1 + 1.2%, respectively, p<0.05, n=5). Conclusions: Cardiac β 1 AR-activated βarrestin2, but not βarrestin1, promotes SERCA2a SUMOylation and activity, increasing cardiac contractility. Given also its anti-inflammatory and anti-apoptotic effects post-MI, cardiac-specific βarrestin2 gene transfer may be a novel and safe inotropic therapy for both acute and chronic HF.

Ultrasound targeted gene therapy in a mouse model of prostate cancer: Evaluation of immune response.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 120-120
Author(s):  
Flavia De Carlo ◽  
Litty Thomas ◽  
Rounak Nande ◽  
Olivia Boskovic ◽  
Gailen Marshall ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gene Transfer ◽  
Cancer Cells ◽  
Transduction Efficiency ◽  
Acquired Immunity ◽  
Prostate Cancer Cells ◽  
Adenoviral Gene Transfer ◽  
Specific Tissue

120 Background: Gene transfer to malignant sites using human adenoviruses (hAd) has been limited because of their immunogenicity. Murine cells often lack some of the receptors needed for hAd infection; therefore, are generally non-permissive for hAd infection and replication, which limits translational studies of adenoviral gene transfer techniques. We developed a gene transfer method, which uses a combination of lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to shield and deliver hAds to a specific tissue bypassing the requirement of the coxsackie and adenovirus receptor (CAR). Methods: Transduction efficiency and GFP protein expression of hAd.GFP was assessed by flow cytometry and fluorescence microscopy in murine TRAMP-C2 and human DU145 prostate cancer cells. Innate and acquired immunity response was determined by ELISA and CTL assay in C57BL/6 mice bearing TRAMP-C2 syngeneic tumor grafts following injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Results: We observed that the murine prostate cancer cells TRAMP-C2 were transduced less efficiently by hAd.GFP than the human DU145 cells. We showed in vitro that the transduction rate was increased significantly in both TRAMP-C2 and DU145 prostate cancer cells when delivering the Ad particles by a combination of MBs and US. Moreover, we observed expression of the GFP transgene in both cell lines at 48 hours and 72 hours. Lack of activation of the innate and acquired immunity was observed in vivo by quantifying IL-6 and TNF-α cytokines, and by assaying neutralizing IgG antibodies and CTLs activity, following intratumoral or intravenous injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Conclusions: This study demonstrates the feasibility of using the TRAMP-C2 murine model of prostate adenocarcinoma to translate our ultrasound-mediated MB-Ad delivery system from the bench to the clinic. Our data provides evidence that the TRAMP-C2 prostate cancer graft model is a suitable system to study in immune competent animals the capacity of lipid-encapsulated perfluorocarbon MBs and US, to shield and deliver hAds to a site-specific tissue bypassing the requirement of specific receptors.

Cardiac contractility modulation by electric currents applied during the refractory period

2002 ◽  
Vol 282 (5) ◽  
pp. H1642-H1647 ◽  
Author(s):  
Satoshi Mohri ◽  
Kun-Lun He ◽  
Marc Dickstein ◽  
Yuval Mika ◽  
Juichiro Shimizu ◽  
...  
Keyword(s):  
Refractory Period ◽  
Cardiac Contractility ◽  
Total Duration ◽  
Peripheral Resistance ◽  
Systolic Pressure ◽  
Segment Length ◽  
Electric Currents ◽  
Inotropic Effects

Inotropic effects of electric currents applied during the refractory period have been reported in cardiac muscle in vitro using voltage-clamp techniques. We investigated how electric currents modulate cardiac contractility in normal canine hearts in vivo. Six dogs were instrumented to measure regional segment length, ventricular volume (sonomicrometry), and ventricular pressure. Cardiac contractility modulating (CCM) electric currents (biphasic square pulses, amplitude ±20 mA, total duration 30 ms) were delivered during the refractory period between pairs of electrodes placed on anterior and posterior walls. CCM significantly increased index of global contractility ( E es) from 5.9 ± 2.9 to 8.3 ± 4.6 mmHg/ml with anterior CCM, from 5.3 ± 1.8 to 8.9 ± 4.0 mmHg/ml with posterior CCM, and from 6.1 ± 2.6 to 11.0 ± 7.0 mmHg/ml with combined CCM ( P < 0.01, no significant change in volume axis intercept). End-systolic pressure-segment length relations showed contractility enhancement near CCM delivery sites, but not remotely. Relaxation was not influenced. CCM increased mean aortic pressure, but did not change peripheral resistance. Locally applied electrical currents enhanced global cardiac contractility via regional changes in myocardial contractility without impairing relaxation in situ.

scholarly journals Improvement in Adenoviral Gene Transfer Efficiency after Preincubation at +37°C in Vitro and in Vivo

2002 ◽  
Vol 5 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Maija Kossila ◽  
Suvi Jauhiainen ◽  
Mikko O. Laukkanen ◽  
Pauliina Lehtolainen ◽  
Maiju Jääskeläinen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transfer Efficiency ◽  
Adenoviral Gene Transfer ◽  
Gene Transfer Efficiency

Adenovirus-mediated gene transfer of hIGF-IB in mouse lungs induced prolonged inflammation but no fibroproliferation

2010 ◽  
Vol 298 (4) ◽  
pp. L492-L500 ◽  
Author(s):  
Caroline Léger ◽  
Ai Ni ◽  
Graciela Andonegui ◽  
Josée Wong ◽  
Connie Mowat ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Gene Transfer ◽  
Cell Activation ◽  
Mesenchymal Cell ◽  
Neutrophil Infiltration ◽  
Adenoviral Gene Transfer ◽  
Matrix Deposition ◽  
End Stage

Pulmonary fibrosis (PF), the end stage of a variety of fibroproliferative lung diseases, is characterized by excessive lung mesenchymal cell activation and extracellular matrix deposition. Most PF is induced after repetitive or chronic lung inflammation; however, a significant portion of PF occurs without apparent inflammation. The mechanisms of fibroproliferation are poorly understood. Studies have shown that cytokines regulating inflammation and tissue repair processes play essential roles in the development of PF. Insulin-like growth factor I (IGF-I) has been shown to stimulate lung mesenchymal cell proliferation and extracellular matrix synthesis in vitro and is significantly elevated in patients with PF. In this study, we investigated whether human IGF-IB (hIGF-IB) expression in the lungs induces PF in a C57BL/6 mouse model. Mice were subjected to adenoviral gene transfer, and the effects of hIGF-IB expression on the lungs were examined 3, 7, 14, 21, and 42 days after gene delivery. hIGF-IB expression induced significant and prolonged inflammatory cell infiltration into the lungs, with an early neutrophil infiltration followed by a late macrophage infiltration. No significant fibroblast or matrix accumulation could be detected in the lungs of these mice. No significant collagen accumulation could be detected in vivo, despite in vitro evidence that hIGF-IB induces collagen mRNA expression in fibroblasts. Therefore, IGF-IB alone is not sufficient to induce fibrosis, and it is possible that a coactivator is required to induce significant fibroproliferation in vivo.

Vascular interleukin-10 protects against LPS-induced vasomotor dysfunction

2005 ◽  
Vol 289 (2) ◽  
pp. H624-H630 ◽  
Author(s):  
Carol A. Gunnett ◽  
Donald D. Lund ◽  
Frank M. Faraci ◽  
Donald D. Heistad
Keyword(s):  
Gene Transfer ◽  
Vascular Dysfunction ◽  
Interleukin 10 ◽  
Wild Type ◽  
Low Concentration ◽  
Adenoviral Gene Transfer ◽  
Vasomotor Dysfunction ◽  
Deficient Mice

We tested the hypotheses that 1) systemic IL-10, after adenoviral gene transfer, protects arteries from impaired relaxation produced by LPS; 2) local expression of IL-10 within the arterial wall protects against vasomotor dysfunction after LPS; and 3) IL-10 protects against vascular dysfunction mediated by inducible NO synthase (iNOS) after LPS. In IL-10-deficient (IL-10−/−) and wild-type (WT, IL-10+/+) mice, LPS in vivo impaired relaxation of arteries to acetylcholine and gene transfer of IL-10 improved responses to acetylcholine. Superoxide levels were elevated in arteries after LPS, and increased levels of superoxide were prevented by gene transfer of IL-10. In arteries incubated with a low concentration of LPS in vitro to eliminate systemic effects of LPS and IL-10 from nonvascular sources, responses to acetylcholine were impaired in IL-10-deficient mice and impairment was largely prevented by gene transfer in vitro of IL-10. In arteries from WT mice in vitro, the low concentration of LPS did not impair responses to acetylcholine. Thus IL-10 within the vessel wall protects against LPS-induced dysfunction. In IL-10-deficient mice, aminoguanidine, which inhibits iNOS, protected against vasomotor dysfunction after LPS. In arteries from iNOS-deficient mice, LPS did not impair responses to acetylcholine. These findings suggest that both systemic and local effects of IL-10 provide important protection of arteries against an inflammatory stimulus and that IL-10 decreases iNOS-mediated impairment of vasorelaxation after LPS.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

scholarly journals An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2694-2700 ◽  
Author(s):  
DR Rill ◽  
RC Moen ◽  
M Buschle ◽  
C Bartholomew ◽  
NK Foreman ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Marrow Transplantation ◽  
Residual Disease ◽  
Marker Gene ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Disease Recurrence ◽  
Malignant Cells

Abstract Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested “remission” marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.

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