Carmela Zincarelli

Analysis of AAV Serotypes 1–9 Mediated Gene Expression and Tropism in Mice After Systemic Injection

This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.

Myocardial Adeno-Associated Virus Serotype 6–βARKct Gene Therapy Improves Cardiac Function and Normalizes the Neurohormonal Axis in Chronic Heart Failure

Background— The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional &bgr;-adrenergic receptor (&bgr;AR) signaling and cardiac function. The peptide &bgr;ARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve &bgr;AR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term &bgr;ARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results— In HF rats, we delivered &bgr;ARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the &bgr;-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. &bgr;ARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac &bgr;AR signaling. Addition of metoprolol neither enhanced nor decreased &bgr;ARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions— Long-term cardiac AAV6-&bgr;ARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, &bgr;ARKct alone improves outcomes more than a &bgr;-blocker alone, whereas both treatments are compatible. These findings show that &bgr;ARKct gene therapy can be of long-term therapeutic value in HF.

An adrenal β-arrestin 1-mediated signaling pathway underlies angiotensin II-induced aldosterone production in vitro and in vivo

Aldosterone produces a multitude of effects in vivo, including promotion of postmyocardial infarction adverse cardiac remodeling and heart failure progression. It is produced and secreted by the adrenocortical zona glomerulosa (AZG) cells after angiotensin II (AngII) activation of AngII type 1 receptors (AT1Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of Gq/11-proteins, to which the AT1R normally couples. Here, we describe a novel signaling pathway underlying this AT1R-dependent aldosterone production mediated by β-arrestin-1 (βarr1), a universal heptahelical receptor adapter/scaffolding protein. This pathway results in sustained ERK activation and subsequent up-regulation of steroidogenic acute regulatory protein, a steroid transport protein regulating aldosterone biosynthesis in AZG cells. Also, this βarr1-mediated pathway appears capable of promoting aldosterone turnover independently of G protein activation, because treatment of AZG cells with SII, an AngII analog that induces βarr, but not G protein coupling to the AT1R, recapitulates the effects of AngII on aldosterone production and secretion. In vivo, increased adrenal βarr1 activity, by means of adrenal-targeted adenoviral-mediated gene delivery of a βarr1 transgene, resulted in a marked elevation of circulating aldosterone levels in otherwise normal animals, suggesting that this adrenocortical βarr1-mediated signaling pathway is operative, and promotes aldosterone production and secretion in vivo, as well. Thus, inhibition of adrenal βarr1 activity on AT1Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.

Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure.

Exercise training has been reported to exert beneficial effects on cardiac function and to reduce morbidity and mortality of chronic heart failure (HF). Augmented sympathetic nervous system (SNS) activity, leading to elevated circulating catecholamine (CA) levels, is a hallmark of chronic HF that significantly aggravates this disease. Exercise training has been shown to also reduce SNS overactivity in HF, but the underlying molecular mechanism(s) remain unidentified. We recently reported that adrenal G protein-coupled receptor kinase-2 (GRK2), an enzyme that regulates the sympathoinhibitory alpha(2)-adrenoceptors (alpha(2)-ARs) present in the CA-producing adrenal medulla, is upregulated in HF, contributing to the chronically elevated CA levels and SNS activity of the disease. In the present study, we tested whether exercise training can affect the adrenal GRK2-alpha(2)-AR-CA production system in the context of HF. For this purpose, a 10-wk-long exercise training regimen of adult male Sprague-Dawley rats starting at 4 wk postmyocardial infarction (post-MI) was employed, and examination at the end of this treatment period revealed significant amelioration of beta-AR-stimulated contractility in response to exercise training, accompanied by cardiac GRK2 reduction and restoration of circulating plasma CA levels. Importantly, adrenal GRK2 expression (72 + or - 5% reduction vs. post-MI untrained) and alpha(2)-AR number were also restored after exercise training in post-MI animals. These results suggest that exercise training restores the adrenal GRK2-alpha(2)-AR-CA production axis, and this might be part of the mechanism whereby this therapeutic modality normalizes sympathetic overdrive and impedes worsening of the failing heart.

Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity

We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

Targeting the β-Adrenergic Receptor System Through G-Protein–Coupled Receptor Kinase 2: A New Paradigm for Therapy and Prognostic Evaluation in Heart Failure From Bench to Bedside

G-protein–coupled receptors (GPCRs) are a superfamily of more than 1000 membrane proteins that respond to a wide spectrum of extracellular signals, modulating various physiopathological processes.1,2 Several GPCRs, such as adrenergic, angiotensin, endothelin, and adenosine receptors, are expressed in cardiovascular (CV) tissues to maintain CV homeostasis. Importantly, GPCR-mediated adrenergic deregulation has been shown to both cause and contribute to the onset and progression of major CV diseases ultimately leading to heart failure (HF). Thus, GPCRs have become salient targets of current pharmacotherapy in CV disorders, and in past decades, many efforts have been made to better clarify their role in the pathophysiology of cardiac disease, focusing not only on receptor functions but also on postreceptor components that mediate or regulate their responses. Among the latter, a relevant role has been attributed to G-protein–coupled receptor kinases (GRKs). In this review, we focus on GRK2, the most abundant and versatile GRK expressed on CV system, tracing the way from initial experimental evidence to more recent data suggesting a potential role for this kinase in the clinical management of HF.1,2 ### GPCR Signaling and GRK Functions: Pathophysiological Background On stimulation, GPCRs interact with heterotrimeric G proteins, which in turn dissociate into 2 functional monomers, namely Gα and Gβγ, both of which modulate different effector systems. Agonist binding to GPCR promotes the activation of complex regulatory mechanisms to protect the receptor from both acute and chronic stimulation, a process termed desensitization. As extensively described, GPCR desensitization involves 3 main events in chronological order: receptor phosphorylation and uncoupling from G proteins, internalization of membrane-bound receptors, and downregulation through reduced mRNA and protein synthesis or increased degradation of internalized receptors.1,2 The desensitization process is mediated by 3 families of proteins: second-messenger–dependent protein kinases, GRKs and arrestins. The defining feature of GRKs is that …

Blockade of β-adrenoceptors restores the GRK2-mediated adrenal α2-adrenoceptor–catecholamine production axis in heart failure

BACKGROUND AND PURPOSE Sympathetic nervous system (SNS) hyperactivity is characteristic of chronic heart failure (HF) and significantly worsens prognosis. The success of β‐adrenoceptor antagonist (β‐blockers) therapy in HF is primarily attributed to protection of the heart from the noxious effects of augmented catecholamine levels. β‐Blockers have been shown to reduce SNS hyperactivity in HF, but the underlying molecular mechanisms are not understood. The GPCR kinase‐2 (GRK2)–α2adrenoceptor–catecholamine production axis is up‐regulated in the adrenal medulla during HF causing α2‐adrenoceptor dysfunction and elevated catecholamine levels. Here, we sought to investigate if β‐blocker treatment in HF could lower SNS activation by directly altering adrenal GRK2 levels.

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density.

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.

Vascular Endothelial Growth Factor Blockade Prevents the Beneficial Effects of β-Blocker Therapy on Cardiac Function, Angiogenesis, and Remodeling in Heart Failure

Background—Impaired angiogenesis in the post-myocardial infarction heart contributes to the progression to heart failure. The inhibition of vascular endothelial growth factor (VEGF) signaling has been shown to be crucial for the transition from compensatory hypertrophy to cardiac failure. Importantly, &bgr;-adrenergic receptor blocker therapy has been also shown to improve myocardial perfusion by enhancing neoangiogenesis in the failing heart. Methods and Results—Eight weeks from surgically induced myocardial infarction, heart failure rats were randomized to receive bisoprolol (B) or vehicle. At the end of a 10-week treatment period, echocardiography revealed reduced cardiac diameters and improved cardiac function in B-treated compared with vehicle-treated rats. Moreover, B treatment was associated with increased cardiac angiogenesis and in vivo coronary perfusion and reduced cardiac fibrosis. Importantly, 2 weeks after B treatment was started, increased cardiac VEGF expression and Akt and endothelial NO synthase activation were observed by comparing B-treated with drug-untreated failing hearts. To test whether the proangiogenic effects of B act via activation of VEGF pathway, rats were intravenously injected with adenoviral vector encoding a decoy VEGF receptor (Ad-Flk) or a control adenovirus (Ad-C), at the start of the treatment with B. After 10 weeks, histological analysis revealed reduced capillary and coronary perfusion in B-treated plus Ad-Flk rats compared with B-treated plus Ad-C rats. Moreover, VEGF inhibition counteracted the positive effects of B on cardiac function and remodeling. Conclusions—&bgr;-Blockade promotes cardiac angiogenesis in heart failure via activation of VEGF signaling pathway. &bgr;-Blocker–induced enhancement of cardiac angiogenesis is essential for the favorable effects of this therapy on cardiac function and remodeling.

Lymphocyte G-protein-coupled receptor kinase-2 is upregulated in patients with Alzheimer's disease.

Alterations in signal transduction pathway of G-protein-coupled receptors (GPCRs) have been found in the cerebrocortex and in the peripheral cultured tissues of patients with Alzheimers disease (AD). The G-protein-coupled receptor kinase-2 (GRK2) plays an important role in regulating the GPCRs signaling: its increased expression is associated with receptor desensitization. The aim of this study was to explore GRK2 levels in peripheral lymphocytes of AD patients and to establish a correlation between lymphocyte protein concentrations and the degree of cognitive impairment. GRK2 mRNA and protein expression were evaluated in the lymphocytes of AD patients with mild or moderate/severe cognitive impairment and in age-matched healthy subjects. Both GRK2 mRNA and protein expression were higher in AD patients lymphocytes compared to controls. Furthermore, lymphocyte GRK2 levels were significantly correlated to the degree of cognitive decline. Our preliminary data suggest that GRK2 is involved in GPCRs coupling dysfunction observed in AD patients. Further studies are needed in order to verify whether the lymphocyte GRK2 might be utilized as a novel biomarker in AD diagnosis and clinical monitoring.