Katrina H. Wilson

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary β2-adrenergic receptor gene delivery

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

α2-Adrenergic receptors in human spinal cord : specific localized expression of mRNA encoding α2-adrenergic receptor subtypes at four distinct levels

Abstract α2-Adrenergic receptor (AR) subtype mRNA (α2a, α2b, α2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing α2AR subtype specific mRNA. α2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to α2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate α2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where α2ARs have not been described. Previously unreported heterogeneity in α2AR subtype distribution (α2a and α2bAR mRNA present, α2cAR mRNA virtually absent) is found at all sites of α2AR mRNA expression in human spinal cord, including locations known to mediate effects of α2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of α2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of α2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

Pharmacology of tamsulosin: Saturation-binding isotherms and competition analysis using cloned α1-adrenergic receptor subtypes

α1‐adrenergic receptors (α1 ARs) are important in the dynamic component of benign prostatic hyperplasia (BPH). Currently, several α1AR antagonists are being used in the treatment of BPH.

Cardiac Gene Delivery With Cardiopulmonary Bypass

Background—Cardiac gene therapy offers the possibility of enhancing myocardial performance in the compromised heart. However, current gene delivery techniques have limited myocardial transgene expression and pose the risk of extracardiac expression. Isolation of the coronary circulation during cardiac surgery may allow for more efficient and cardiac-selective gene delivery in a clinically relevant model. Methods and Results—Neonatal piglets (3 kg) underwent a median sternotomy and cardiopulmonary bypass, followed by aortic cross-clamping with 30 minutes of cardioplegic arrest. Adenoviral vectors containing transgenes for either &bgr;-galactosidase (adeno-&bgr;-gal, n=11) or the human &bgr;2-adrenergic receptor (adeno-&bgr;2-AR, n=15) were administered through the cardioplegia cannula immediately after arrest and were allowed to dwell in the coronary circulation during the cross-clamp period. After 1 week, the animals were killed, and their heart, lungs, and liver were excised and examined for gene expression. Analysis of &bgr;-galactosidase staining revealed transmural myocardial gene expression among animals receiving adeno-&bgr;-gal. No marker gene expression was detected in liver or lung tissue. &bgr;-AR density in the left ventricle after adeno-&bgr;2-AR delivery was 396±85% of levels in control animals (P <0.01). Animals receiving adeno-&bgr;2-AR and control animals demonstrated similar &bgr;-AR density in both the liver (114±8% versus 100±9%, P =NS) and lung (114±7% versus 100±9%, P =NS). There was no evidence of cardiac inflammation. Conclusions—By using cardiopulmonary bypass and cardioplegic arrest, intracoronary delivery of adenoviral vectors resulted in efficient myocardial uptake and expression. Undetectable transgene expression in liver or lung tissue suggests cardiac-selective expression.

Myocardial Gene Transfer and Overexpression of β2-Adrenergic Receptors Potentiates the Functional Recovery of Unloaded Failing Hearts

Background—Mechanical assistance of the failing left ventricle (LV) can lead to functional recovery after a period of unloading, including restoration of &bgr;-adrenergic receptor (&bgr;AR) inotropic reserve. We tested whether prolonged LV unloading of failing rabbit hearts by use of a heterotopic transplantation technique could lead to recovery and whether adenoviral gene transfer of a &bgr;2AR transgene (Adv-&bgr;2AR) could alter this process. Methods and Results—Heart failure was induced by coronary artery ligation in adult New Zealand White rabbits. After 4 weeks, failing hearts were heterotopically transplanted into recipient rabbits, allowing normal coronary perfusion but complete LV unloading. We also placed an LV latex balloon for remote access and in vivo physiological analysis. We found that there was reversal of signaling and functional abnormalities after 30 days of unloading. In another set of failing hearts, we randomly delivered, at the time of transplantation, either 2×1011 viral particles of Adv-&bgr;2AR or saline via the coronary arteries. Sham-operated animals with nonfailing hearts served as controls. After 5 days of unloading, in vivo LV contractility (LV dP/dtmax) and relaxation (LV dP/dtmin) were significantly decreased in saline-treated failing hearts compared with control nonfailing hearts (P <0.05). In failing hearts treated with Adv-&bgr;2AR, however, LV dP/dtmax and LV dP/dtmin were improved in response to higher preloads (P <0.05) and &bgr;AR stimulation (P <0.01). Conclusions—Heterotopic transplantation in the rabbit does allow recovery of the failing heart, and &bgr;2AR overexpression acutely enhances this functional improvement. Accordingly, genetic manipulation of &bgr;AR signaling may represent a novel molecular adjunct to mechanical assistance to facilitate functional myocardial recovery.

Adenovirus-mediated gene transfer of the β2-adrenergic receptor to donor hearts enhances cardiac function

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of β2-adrenergic receptors (β2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human β2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 × 1010 p.f.u. of adenovirus encoding the β2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the β2-AR agonist, zinterol. Treatment with the β2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean β-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 ± 286 versus β2-AR, 6250.6* ± 432.5 mmHg/s; n = 10, *P < 0.02). β2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of β2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.

Catheter-based intracoronary myocardial adenoviral gene delivery: Importance of intraluminal seal and infusion flow rate

Although percutaneous, adenoviral-mediated intracoronary gene delivery to the heart has been demonstrated in some species, consistent and safe methodology is needed before clinical applicability is possible. In this study, we examine the effects of altering intracoronary flow rate and obtaining an adequate seal between the catheter and the coronary lumen on successful cardiac gene delivery and myocardial injury in both piglets and adult rabbits. To study the efficacy of in vivo myocardial gene transfer, we utilized adenoviral vectors containing either the beta(2)-adrenergic receptor or beta-galactosidase. The left circumflex coronary artery of piglets and the right coronary artery of rabbits were catheterized under fluoroscopic guidance and adenovirus solutions were injected using varying flow rates with or without balloon inflation. Successful transgene delivery to the heart was determined approximately 1 week after coronary infusions. Histologic analysis was also performed in all animals to determine the extent of myocardial injury. Our results indicate that efficient and reproducible cardiac transgene expression utilizing intracoronary delivery is dependent upon the infusion flow rate and, in larger animals, requires an intraluminal seal. Excessive flow rate is associated with greater myocardial injury. Thus, conditions can be established and controlled to improve future investigational and clinical application of catheter-based intracoronary myocardial gene therapy.

Adenoviral gene transfer to the heart during cardiopulmonary bypass: effect of myocardial protection technique on transgene expression

OBJECTIVE Adenoviral gene transfer to the arrested heart during cardiopulmonary bypass (CPB) is a novel method of allowing prolonged vector contact with the myocardium. In this model we investigated the importance of temperature, duration of arrest and cardioplegia on transgene expression. METHODS First-generation adenoviral vector (1 x 10(12) total viral particles) containing the transgene for the human beta2-adrenoceptor (Adeno-beta(2)AR) or beta-galactosidase (Adeno-beta(gal)) was delivered to neonatal piglets via the proximal aorta, during simulated cardiac surgery, and allowed to dwell for the cross-clamp duration. Four treatment groups received Adeno-beta(2)AR. Groups A (n=4) and B (n=6) underwent cold crystalloid cardioplegia arrest for 10 and 30 min, respectively, Group C (n=5) underwent warm crystalloid cardioplegia arrest for 10 min, and Group D (n=5) underwent warm fibrillatory arrest for 10 min. Group E (n=6) received Adeno-beta(gal) and underwent cold crystalloid cardioplegia arrest (30 min). Animals were weaned off CPB and recovered for 2 days. Receptor density was assessed in membrane fractions using radioligand binding and compared using the Mann-Whitney U-test. RESULTS Left ventricular transgene overexpression, as evidenced by elevated betaAR density, following Adeno-beta(2)AR treatment was greatest with cold cardioplegia (Group A 588+/-288.8 fmol/mg; P=0.002 and Group B 520+/-250.9 fmol/mg; P=0.01) versus control (Group E 109+/-8.4 fmol/mg). Overexpression also occurred with warm cardioplegia (Group C 274+/-69.5 fmol/mg; P=0.05) and ventricular fibrillation (Group D 215+/-48.4 fmol/mg; P=0.02) versus control. Comparison of the combined cold cardioplegia groups versus those treated with warm conditions showed a trend towards increased expression with cold conditions (P=0.1). Receptor density was also significantly increased in the right ventricle of animals in Group B (165+/-18.1 fmol/mg; P=0.03) and Group D (181+/-23.4 fmol/mg; P=0.02) versus control (Group E 118+/-5.8 fmol/mg). CONCLUSIONS Cold crystalloid cardioplegia is not detrimental to gene transfer in vivo. In fact, there was a trend towards increased left ventricular transgene expression when the adenoviral vector was delivered following cold versus warm cardioplegia. Shorter periods of contact with the vector may reduce transgene overexpression. Therefore, gene transfer is possible during cardiac surgery with clinically used myocardial protection techniques.

In situ hybridization: identification of rare mRNAs in human tissues

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Alpha-adrenergic mRNA subtype expression in the human nasal turbinate.

PurposeAlpha-adrenergic receptor (AR) agonist drugs (e.g., epinephrine) are commonly used for upper airway procedures, to shrink the mucosa, retard absorption of local anesthetic agents, and improve visualization by limiting hemorrhage. Decongestant therapy often also includes αAR agonist agents, however overuse of these drugs (e.g., oxymetazoline) can result in chronic rhinitis and rebound increases in nasal secretion. Since current decongestants stimulate αARs non-selectively, characterization of αAR subtype distribution in human airway (nasal turbinate) offers an opportunity to refine therapeutic targets while minimizing side-effects. We, therefore, investigated αAR subtype expression in human nasal turbinate within epithelial, duct, gland, and vessel cells using in situ hybridization.MethodsSince sensitive and specific anti-receptor antibodies and highly selective αAR subtype ligands are currently unavailable, in situ hybridization was performed on sections of three human nasal turbinate samples to identify distribution of αAR subtype mRNA. Subtype specific35S-labelled mRNA probes were incubated with nasal turbinate sections, and protected fragments remaining after RNase treatment analyzed by light and darkfield microscopy.ResultsIn non-vascular tissue αld AR mRNA predominates, whereas notably the α2c is the only αAR subtype present in the sinusoids and arteriovenous anastamoses.ConclusionCombined with the current understanding that AR-mediated constriction of nasal sinusoids underpins decongestant therapies that minimize secretions and shrink tissues for airway procedures, these findings suggest that α2c AR subtypes provide a novel selective target for decongestant therapy in humans.ObjectifLes médicaments agonistes des récepteurs alphaadrénergiques (AR) (par ex., l’épinéphrine) sont communément utilisés lors des interventions sur les voies aériennes supérieures, afin de rétrécir la muqueuse, de retarder l’absorption d’agents anesthésiques locaux et d’améliorer la visualisation en limitant l’hémorragie. Un traitement décongestionnant inclut également souvent des agents agonistes αAR; toutefois, la surutilisation de ces médicaments (par ex., l’oxymétazoline) peut engendrer une rhinite chronique et l’augmentation rebond des sécrétions nasales lors de la cessation du traitement. Puisque les décongestionnants actuels stimulent les αAR de manière non-sélective, la caractérisation de la distribution des sous-types d’αAR dans les voies aériennes de l’homme (cornet nasal) offre la possibilité de perfectionner les cibles thérapeutiques tout en minimisant les effets secondaires. C’est pourquoi nous avons examiné l’expression des sous-types d’αAR au niveau du cornet nasal humain dans les cellules épithéliales, du canal, des glandes et des vaisseaux, à l’aide d’une hybridation in situ.Méthodeétant donné que des anticorps anti-récepteurs sensibles et spécifiques ainsi que des ligands très sélectifs des sous-types d’αAR sont disponibles actuellement, l’hybridation in situ a été effectuée sur des sections de trois échantillons de cornet nasal humain afin d’identifier la distribution d’ARN messager des sous-types d’αAR. Des sondes d’ARN messager marquées au35S et spécifiques au sous-type ont été incubées avec des sections de cornet nasal, et les fragments protégés restants après le traitement à la ribonucléase ont été analysés par microscopie optique et sur fond noir.RésultatsDans les tissus non-vasculaires, l’ARN messager AR αld est prédominant, alors que le α2c est notablement le seul sous-type d’αAR présent dans les sinusoïdes et les anastomoses artérioveineuses.ConclusionOn considère maintenant que la constriction des sinus nasaux médiée par AR est à la base des thérapies de décongestion qui minimisent les sécrétions et rapetissent les tissus lors des interventions sur les voies aériennes. Ces résultats suggèrent donc que les sous-types d’AR α2c fournissent une nouvelle cible sélective pour les traitements de décongestion chez les humains.