Myocardial ?2-adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failuresummarize and critiquing the following scientific paper, in terms of it both its merits and flaws. Marking Scheme: Well organised and well expressed answer which shows very good understanding, evidence of independent study and critical evaluation. Covers all aspects of the subject that could reasonably be expected, accurately and in sufficient detail.Myocardial ?2-adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failureBACKGROUND AND PURPOSEWe investigated whether b2-adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and leftventricular (LV) remodeling of the failing heart.EXPERIMENTAL APPROACHWe explored the angiogenic effects of b2-adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heartfailure (HF). Cardiac adenoviral-mediated b2-adrenoceptor overexpression was obtained via direct intramyocardial injection4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observationto b2-adrenoceptor -/- mice undergoing MI.KEY RESULTSTransgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weekspost-gene delivery. In HF rats, cardiac b2-adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulatedcardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-b2-adrenoceptor HF rats showedimproved LV remodeling and cardiac function. Importantly, b2-adrenoceptor overexpression was associated with a markedlyincreased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At themolecular level, cardiac b2-adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway.In b2-adrenoceptor-/- mice, we found a ~25% reduction in cardiac capillary density compared with b2-adrenoceptor+/+ mice.The lack of b2-adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse globalcardiac contractility compared with controls.CONCLUSIONS AND IMPLICATIONb2-Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOSpathway seems to be strongly involved in this mechanism.AbbreviationsAd, adenovirus; ECs, endothelial cells; eNOS, endothelial NOS; FS, LV fractional shortening; GFP, green fluorescentprotein; GRK2, G-protein coupled receptor kinase-2; HF, heart failure; ICI118551, erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol; ISO, isoprenaline; KO, knockout; LAD, left anterior descending coronary artery; LV, leftventricular; MI, myocardial infarction; SNS, sympathetic nervous systemBJP British Journal ofPharmacologyDOI:10.1111/j.1476-5381.2012.01954.xwww.brjpharmacol.org2348 British Journal of Pharmacology (2012) 166 2348.2361 c 2012 The AuthorsBritish Journal of Pharmacology c 2012 The British Pharmacological SocietyIntroductionHeart failure (HF) is a complex clinical syndrome characterizedby left ventricular (LV) dysfunction accompanied bygeneralized hyperactivation of neurohormonal status (Rengoet al., 2004; Mann and Bristow, 2005; Lymperopoulos et al.,2007a). Although the sympathetic nervous system (SNS)overdrive is beneficial immediately after cardiac injury,aiming to preserve cardiac output, over time, it becomesdetrimental, facilitating the worsening of cardiac contractility.The prolonged and sustained HF-related SNS activationinduces several noxious effects on myocardial and vascularfunction, and is responsible for the dysregulation of cardiacb-adrenoceptors, including b1-adrenoceptor down-regulationand b1- and b2-adrenoceptor uncoupling from signaltransducingG-proteins (Rockman et al., 2002; Lohse et al.,2003; Feldman et al., 2005; Leosco et al., 2007; Triposkiadiset al., 2009), via increased cardiac G-protein coupled receptorkinase-2 (GRK2) expression/activity (Lefkowitz, 1993;Ungerer et al., 1993; 1994; Rengo et al., 2011). The moleculardysfunction of b-adrenoceptor signalling accounts for thereduced inotropic response to adrenergic stimulation in thefailing myocardium.Over the past two decades, several data have beenproduced proving different effects between b1- andb2-adrenoceptor signalling on cardiac physiology and pathophysiology(Lands et al., 1967; Communal et al., 1999; Zaugget al., 2000; Xiao et al., 2004). However, the role ofb2-adrenoceptors in HF has not been completely elucidated(Zhu et al., 2005). Previous evidence indicated that cardiacthe overexpression of b2-adrenoceptors improves contractilityin the healthy heart (Milano et al., 1994; Dorn et al., 1999;Maurice et al., 1999; Liggett et al., 2000), and potentiates thefunctional recovery of unloaded rabbit failing myocardium(Tevaearai et al., 2002). Furthermore, other evidence has beenobtained indicating that pharmacological stimulation ofb2-adrenoceptors in the post-ischaemic failing heart inducescardioprotection preventing maladaptive remodelling, andcurbing HF progression (Ahmet et al., 2004; Xydas et al.,2006).Recently, b2-adrenoceptor signalling has been demonstratedto be involved in angiogenesis regulation. In an experimentalmodel of hindlimb ischaemia, it has beendemonstrated that b2-adrenoceptors play a pivotal role in thecontrol of endothelial cells (ECs) function. Adenoviralmediatedb2-adrenoceptor overexpression in the ischaemichindlimb leads to enhanced EC proliferation and migrationwith the final effect of improved ischaemia-induced angiogenesis(Iaccarino et al., 2005). This pro-angiogenic effectseems to be ascribed to increased b2-adrenoceptor-dependentvascular endothelial growth factor (VEGF) production andrelease (Iaccarino et al., 2005). Moreover, further findings supportingthe important role of b2-adrenoceptors in angiogenesishave been obtained in b2-adrenoceptor knockout micesubjected to chronic hindlimb ischaemia (Ciccarelli et al.,2011). Importantly, the impaired angiogenic response toischaemia, observed in these mice, is restored by intravascularb2-adrenoceptor gene transfer (Ciccarelli et al., 2011).However, whether b2-adrenoceptors regulate angiogenesis inthe heart has never been previously tested either in physiologicalor in pathological conditions. With respect to this,previous findings from our group (Leosco et al., 2008) andothers (Karam et al., 1990) indicate that angiogenic responsesin the failing heart are inadequate and that impairments inthe cardiac capillary and arteriolar network during HF plays akey role in post-ischaemic heart dysfunction. At the molecularlevel, this is associated with a blunted activation of the VEGF/PKB (Akt)/endothelial-NOS (eNOS) pathway. Nevertheless,the angiogenesis impairment can be reverted through interventions,such as exercise training, leading to an improvementin cardiac b-adrenoceptor signalling and reactivation ofthe PKB pro-angiogenic pathway (Leosco et al., 2008).We aimed to evaluate for the first time whether the beneficialeffects of b2-adrenoceptor overexpression in thefailing myocardium may be correlated to the restoration ofHF-related impairment of cardiac angiogenesis. We utilizedan adenoviral-mediated intramyocardial gene delivery technique,which allows the robust expression of the transgene inthe LV (Rengo et al., 2009; Zincarelli et al., 2010). To furthersupport the notion that the b2-adrenoceptor plays a crucialrole in ischaemia-induced angiogenesis, we also studiedthe phenotype of post-myocardial infarction (MI) HFb2-adrenoceptor knockout (KO) mice.MethodsExperimental proceduresThe study protocol was designed in accordance with TheGuide for Care and Use of Laboratory Animals of the NationalInstitutes of Health (NIH Publication No. 85.23, Revised1996), and was approved by the Ethics Committee for the Useof Animals in Research of our Institution. An expandedMethods section appears in the online-only Data Supplement.The results of all studies involving animals are reportedin accordance with the ARRIVE guidelines (Kilkenny et al.,2010; McGrath et al., 2010).For experimental MI induction, rats were anaesthetizedwith 4% isoflurane. Deep anaesthesia was confirmed by thelack of a response to noxious stimuli. Next, rats were intubatedand ventilated with a mixture of O2 and 2% isofluranewith a pressure-controlled ventilator. After thoracotomy, MIwas induced by permanent ligation of the left anteriordescending coronary artery (LAD). Subsequently, chest andskin were closed in layers. Rats were observed until theyawakened, and then returned to the Animal Care Unit. Standardpostoperative care, including analgesic (buprenorphine400 IU?Ekg-1 i.p. 20 min before surgery, ketoprofen 10 mg?Ekg-1s.c. injection for 2 days after surgery), was provided. MI inmice was induced under general anaesthesia with isoflurane(2%), the heart was exposed and temporarily dislocatedvia a small left thoracotomy to place a suture ligation of theLAD (Lymperopoulos et al., 2009; 2010). Myocardial genetransfer in rats was achieved by direct intramyocardial injection4 weeks post-MI (Rengo et al., 2009). Echocardiographicevaluations were performed at 4 weeks post-MI and at the endof the study (Leosco et al., 2008). Basal and isoprenalinestimulatedLV contractility was evaluated by invasive haemodynamicstudies at 2 and 4 weeks after gene delivery. At theend of the study period, animals were killed by cervical dislocationunder deep anaesthesia.b2-adrenoceptor regulates angiogenesis in heart failure BJPBritish Journal of Pharmacology (2012) 166 2348.2361 2349Myocytes contractilityMyocytes were isolated from the non-infarcted zone of the LVby a standard enzymatic digestion (Leosco et al., 2008; Rengoet al., 2009). Contractility was evaluated under baseline andafter agonist-mediated b1-adrenoceptor stimulation by NA at10-7 M or higher concentrations in the presence of thea1-adrenoceptor antagonist, prazosin (10-6 M).Myocardial perfusion studiesMyocardial perfusion was determined using 15 mm fluorescentmicrospheres (Triton Technology, Inc., San Diego, CA,USA). Cardiac and blood samples were processed for microspheredetermination. Myocardial blood flow was measuredat basal and after maximal vasodilatation.HistologyCapillary and arteriolar length densities were evaluated inborder and remote zones of the infarcted area. Capillarieswere detected by Lectin Bandeiraea simplicifolia-I staining.Arterioles were identified by immunofluorescence usinganti-SM a-actin antibody (Leosco et al., 2008).b-Adrenoceptor signallingReceptor binding, adenylyl cyclase activity and GRK2 proteinassays were obtained as previously described (Rengo et al.,2010).VEGF/PKB/eNOS measurementVEGF, PKB, Ser473-phospho-PKB, eNOS and Ser1177-phosphoeNOSprotein levels were performed by Western blot analysis(Lymperopoulos et al., 2007b; 2008).Statistical analysisData are summarized as mean ¨« SEM. Comparisons weremade with the use of Student?fs t-tests or ANOVA as appropriate.A Bonferroni correction was applied to the probabilityvalues whenever multiple comparisons arose. Values of P <0.05 were considered significant.ResultsStudy design and transgenes expressionAt 1 month after MI, a time point with established HF (Leoscoet al., 2008), rats were randomly allocated to three differentgroups receiving cardiac gene transfer of: adenovirus (Ad).b2-adrenoceptor, Ad.green fluorescent protein (GFP), or vehicle(saline). One day before gene delivery, all groups were analysedby echocardiography to confirm the presence of similar levelsof LV dysfunction and HF. All groups were then studied overthe course of 4 more weeks (Figure 1A), and all assays in the 3HF groups were compared with a control sham-operated groupthat received neither MI nor gene transfer (4 experimentalHaemodynamic EchohaemodynamicEcho4 weeksHF Ad-GFP HF Ad-b2-AR HFcontrol2 weeks aftergene delivery4 weeks aftergene deliveryHFAd-??2-AR450 **400350300250200150Total ??AR densitypmol mg.1 of membrane protein10050045403530252015% Green cardiomyocyte10501 day 2 weeks 2 weeksA MI Gene deliveryBCDEFigure 1(A)Overall design of the 8-week study. (B) Representative GFP fluorescence microscopy (left), light microscopy (middle) and overlay of both (right)of LV myocardium 2 weeks after intramyocardial Ad-GFP delivery. (C) b2-Adrenoceptor (AR) immunohistochemistry in LV tissue from Ad-GFP- (left,control) and Ad-b2-adrenoceptor-treated (right) rats (2 weeks post-gene delivery). Magnification 40. (D) Total b-adrenoceptor density (B) incardiac homogenates purified from hearts of HF Ad-b2-adrenoceptor and HF control (HF-saline and HF Ad-GFP) groups at 2 weeks after genetherapy (n = 6 and 8 for each group); *P < 0.001 vs. HF control. (E) Right panel; percentage (%) of GFP-stained isolated myocytes assessed 2 and4 weeks following Ad-GFP in vivo gene delivery to HF rats by direct intra-myocardial injection (n = 5 for each time point). Green myocytes fromeach rat heart were counted in five randomly selected fields and expressed as percentage of the total number of myocytes per field. *P < 0.001vs. 4 weeks after gene delivery. Data are presented as means ¨« SEM. Left panel: representative GFP fluorescence microscopy (upper), lightmicroscopy (middle) and overlay of both (lower) of myocytes 2 weeks after Ad-GFP delivery.BJP G Rengo et al.2350 British Journal of Pharmacology (2012) 166 2348.2361groups in total). At 2 weeks post-gene delivery, both transgenes(b2-adrenoceptor and GFP) were robustly expressed in the LVofthe respective groups. GFP fluorescence in cardiac sectionsfrom Ad-GFP-treated rats confirmed that a large area of the LVfree wall was transduced, although the expression was nothomogeneous (Figure 1B). b2-Adrenoceptor immunohistochemistryof cardiac sections from Ad-b2-adrenoceptor-treatedHF rats showed comparable areas of the LV transduced(Figure 1C), and the b-adrenoceptor binding experimentshowing a 15-fold increase in membrane receptors comparedwith HF-saline and HF-GFP hearts (Figure 1D). Moreover, toconfirm that our gene delivery technique supported efficientcardiac expression in vivo, 2 and 4 weeks after gene delivery ofAd-GFP, GFP expression was evaluated by detection of greenfluorescence in cardiomyocytes isolated from the LV as previouslydescribed (Rengo et al., 2009). Interestingly, at 2 weeksafter Ad-GFP gene delivery we found a transduction efficiencyto the LV that was >35% of total isolated LV myocytes,whereas, at 4 weeks post-gene delivery, GFP expression wasminimal (less than 5%) (Figure 1E), which is consistent withthe duration of in vivo expression seen by us and others usingAd (Maurice et al., 1999).In vivo effects of cardiac b2-adrenoceptorsoverexpression on LV remodelling andcardiac contractilityAt 1 month post-MI and before gene delivery, all HF groupshad significantly impaired cardiac function compared withsham rats. As assessed by echocardiography, LV fractionalshortening (FS) and internal diameter at diastole were comparablein all the HF groups, indicating a similar degree ofcardiac dysfunction (Table 1).At 2 weeks post-gene delivery, as assessed by in vivo invasivehaemodynamic analysis, HF control groups (HF-Salineand HF-GFP) showed impaired basal and isoprenaline (ISO)-stimulated cardiac contractility, and increased LV enddiastolicpressure compared with sham, as expected(Figure 2). Two weeks of b2-adrenoceptor overexpressionresulted in enhanced basal cardiac contractility and reducedLV end-diastolic pressure compared with HF-saline and -GFPrats (Figure 2 and Table S1).At 4 weeks from gene transfer, a time point whenadenoviral-mediated b2-adrenoceptor overexpression wasminimal, the beneficial effects of b2-adrenoceptor genedelivery on basal cardiac contractility disappeared, butb-adrenoceptor mediated contractility induced by ISO wassignificantly increased compared with HF controls (Table 2).Interestingly, NA was able to increase b1-adrenoceptordependentcontractility in cardiomyocytes extracted fromAdV-b2-adrenoceptor infected hearts compared with HFcontrol. This result indirectly shows there is no obliterationof b1-adrenoceptor responses in isolated cells (Figure S1). Atthe end of the study-period, HF-saline and HF-GFP ratsshowed further deterioration of cardiac function and progressionof LV maladaptive remodelling (LV dilatation andreduced LVW/BW) compared with the echocardiographicTable 1Physical and echocardiographic data in sham-operated and HF rats at 4 weeks after myocardial infarctionSham HF saline HF Ad-GFP HF Ad-b2-adrenoceptorPhysical dataBody wt (kg) 0.408 ¨« 0.017 0.372 ¨« 0.012* 0.360 ¨« 0.018* 0.366 ¨« 0.010*Heart wt (g) 1.15 ¨« 0.09 2.22 ¨« 0.21* 2.30 ¨« 0.19* 2.16 ¨« 0.26*Heart wt/body wt (g?Ekg-1) 2.86 ¨« 0.13 5.94 ¨« 0.63* 6.40 ¨« 0.56* 5.89 ¨« 0.60*Lung wt (g) 1.68 ¨« 0.18 2.87 ¨« 0.22* 3.0 ¨« 0.38* 2.92 ¨« 0.45*Lung wt/body wt (g?Ekg-1) 4.06 ¨« 0.34 7.70 ¨« 0.85* 8.3 ¨« 0.92* 7.88 ¨« 0.67*Echocardiographic dataHeart rate (beats min-1) 366 ¨« 22 355 ¨« 16 348 ¨« 32 352 ¨« 28LV internal diameter (mm)Diastolic 5.72 ¨« 0.45 8.86 ¨« 0.74* 9.12 ¨« 0.82* 9.36 ¨« 1.0*Systolic 3.05 ¨« 0.28 6.35 ¨« 0.62* 6.22 ¨« 0.74* 6.40 ¨« 0.82*LV FS (%) 46.4 ¨« 6.0 27.8 ¨« 5.2* 30 ¨« 4.7* 29.2 ¨« 5.6*Interventricular septum (mm)Diastolic 1.49 ¨« 0.9 1.03 ¨« 0.11* 1.00 ¨« 0.8* 1.04 ¨« 0.9*Systolic 2.64 ¨« 0.32 1.06 ¨« 0.12* 1.03 ¨« 0.6* 1.08 ¨« 0.10*LV posterior wall (mm)Diastolic 1.53 ¨« 0.17 1.88 ¨« 0.18* 1.86 ¨« 0.13* 1.85 ¨« 0.17*Systolic 2.49 ¨« 0.30 2.57 ¨« 0.26 2.55 ¨« 0.15 2.60 ¨« 0.18Sham, n = 10; HF-saline, n = 12; HF Ad-GFP, n = 11; HF Ad-b2-adrenoceptor, n = 12.*P < 0.05 vs Sham.HF, heart failure; LV, left ventricle; FS, fractional shortening.b2-adrenoceptor regulates angiogenesis in heart failure BJPBritish Journal of Pharmacology (2012) 166 2348.2361 2351measurements performed at 1-month post-MI, as expected(Table 1, Table 2 and Figure 3). Importantly, b2-adrenoceptoroverexpression resulted in improved FS, reduced ventricularsystolic and diastolic diameters, and increased LVW/BW comparedwith HF control groups (Table 2 and Figure 3). Finally,at the post-study analysis of LV infarct size, we found similarinfarct sizes among all study groups (Table 2). In this regard,the lack of a reduction in infarct size in b2-adrenoceptoroverexpressing HF rats should be ascribed to the fact thatgene delivery was performed late after MI when infarcthealing was complete.Taken together, these data indicate that b2-adrenoceptoroverexpression exerts a favourable effect on post-MI LV contractilityand remodelling. Interestingly, the positive effectson isoprenaline-stimulated LV contractility and on adaptivehypertrophic remodelling also persisted when the transgeneexpression was exhausted.Effects of b2-adrenoceptors overexpressionon cardiac angiogenesis and myocardialblood flowAt 2 weeks after gene delivery, capillary density, but notarteriolar length, was significantly improved in HF-b2-adrenoceptor rats compared with the HF-saline and HF-GFPgroups (Supplemental Table 1).Figure 4 displays histological and blood flow data in allstudy groups at 4 weeks after gene delivery. All HF groupsshowed a significant reduction in both capillary density andarteriolar length compared with sham animals. Interestingly,b2-adrenoceptor overexpression induced a significant reactivationof the angiogenic mechanims in HF hearts. In fact,capillary and arteriolar density in both border and remotezones of the LV were significantly increased in the HF Ad-b2-adrenoceptor group compared with HF-saline and HF-GFP20 000ABC18 00016 00014 00012 00010 0008000LV . dP/dt (mmHg?Es.1) LV + dP/dt (mmHg?Es.1)60004000200000.1000.2000.3000.4000.5000.6000.7000.8000.9000.10 000C 0.1***** **** **?æ*?æ*?æ*?æ*?æ*?æ*?æ*?æ* ?æ0.5ISO (ug?Ekg.1?Emin.1)1.0C 0.1 0.5051015LVEDP mmHg20251.0Sham HFsalineHFAd-GFPHFAd-??2-ARShamHF salineHF AD-GFPHF AD-??2ARFigure 2(A) Average LV +dP/dt and LV -dP/dt values (B) at 2 weeks post-gene therapy in the four experimental groups evaluated under basal conditionsand after isoprenaline stimulation. (C) Average left ventricle end diastolic pressure (LVEDP) in the four experimental groups. Sham, n = 11;HF-saline, n = 13; HF Ad-GFP, n = 12; HF Ad- b2-adrenoceptor (AR), n = 12. ANOVA analysis and Bonferroni test were used among all groups. Dataare presented as mean ¨« SEM. *P < 0.05 vs sham at basal or at each respective dose of isoprenaline; ?æP < 0.05 vs HF-saline and HF Ad-GFP at basalor at each respective dose of isoprenaline.BJP G Rengo et al.2352 British Journal of Pharmacology (2012) 166 2348.2361hearts. Importantly, in HF Ad-b2-adrenoceptor rats, weobserved a further increase in capillary density between 2 and4 weeks post-gene delivery (Figure 4 and SupplementalTable 1). Moreover, although myocardial blood flow andcoronary reserve were significantly reduced in all HF groupscompared with sham, b2-adrenoceptor overexpressionresulted in a significant improvement in both perfusionparameters compared with HF-controls (Figure 4).Myocardial b-adrenoceptor status after invivo gene deliveryTwo weeks after gene therapy, cardiac b-adrenoceptor densitywas significantly reduced in HF controls compared withsham, while the HF Ad-b2-adrenoceptor group showed a15-fold increase in total membrane b-adrenoceptors comparedwith HF controls, proving the efficacy of the viralmediatedgene delivery technique (Figure 1 and Table 3).b2-Adrenoceptor overexpression was accompanied by higherbasal cardiac cAMP levels compared with HF-saline andHF-GFP groups. As expected, in HF control groups, cAMPcardiac levels were lower compared with sham (Table 3). ISOinduced roughly 2- to 2.5-fold increases in adenylate cyclaseactivity in all HF groups. Myocardial GRK2 levels, measuredby Western blotting, were significantly up-regulated inHF-saline and GFP groups compared with sham. Inb2-adrenoceptor-infected hearts, cardiac GRK2 expression wasdecreased almost to the sham levels.Interestingly, at 4 weeks post-gene delivery, a time pointwhen adenoviral-mediated overexpression of transgenes wasminimal, total b-adrenoceptor density and cAMP productionremained still higher in Ad-b2-adrenoceptor-infected heartscompared with HF controls (Table 3). Accordingly, cardiacGRK2 protein expression was still significantly lower inTable 2Physical, haemodynamic and echocardiographic parameters at 4 weeks after cardiac gene deliverySham-operated HF Saline HF Ad-GFP HF Ad-b2-adrenoceptorPhysical dataBody wt (kg) 0.426 ¨« 0.024 0.388 ¨« 0.022* 0.390 ¨« 0.019* 0.382 ¨« 0.028*Heart wt (g) 1.17 ¨« 0.07 2.58 ¨« 0.28* 2.42 ¨« 0.19* 2.82 ¨« 0.23*?æHeart wt/body wt (g?Ekg-1) 2.75 ¨« 0.14 6.66 ¨« 0.37* 6.12 ¨« 0.23* 7.36 ¨« 0.34*?æLung wt (g) 1.70 ¨« 0.09 3.15 ¨« 0.30* 3.22 ¨« 0.16* 2.14 ¨« 0.19*?æLung wt/body wt (g?Ekg-1) 3.98 ¨« 0.32 8.22 ¨« 0.46* 8.27 ¨« 0.38* 5.60 ¨« 0.36*?æHemodynamic and echo dataHeart rate (beats min-1) 355 ¨« 32 342 ¨« 28 340 ¨« 29 365 ¨« 26LVSP (mmHg) 136 ¨« 12 111 ¨« 10* 109 ¨« 8* 123 ¨« 9*?æLVEDP (mmHg) 4.5 ¨« 0.8 21 ¨« 5* 20 ¨« 6** 10 ¨« 3*?æLV +dP/dt (mmHg?Es-1)Baseline 8842 ¨« 457 4053 ¨« 268* 4238 ¨« 224* 4427 ¨« 313*ISO (1 mg?Ekg-1?Emin-1) 16 724 ¨« 966 6558 ¨« 236* 6886 ¨« 287* 9569 ¨« 461*?æLV .dP/dt (mmHg?Es-1) -6 488 ¨« 440 -3153 ¨« 235* -2923 ¨« 321* -3366 ¨« 357*Baseline -8 421 ¨« 654 -4115 ¨« 586* -3934 ¨« 443* -5340 ¨« 523*?æISO (1 mg?Ekg-1?Emin-1)LV internal diameter (mm)Diastolic 5.69 ¨« 0.37 9.52 ¨« 0.48* 9.43 ¨« 0.56* 7.10 ¨« 0.32*?æSystolic 3.10 ¨« 0.22 7.22 ¨« 0.39* 7.29 ¨« 0.44* 5.10 ¨« 0.36*?æLV fractional shortening (%) 45.5 ¨« 8 24.1 ¨« 4.6* 22.8 ¨« 5.9* 34.6 ¨« 7.2*?æInterventricular septum (mm)Diastolic 1.47 ¨« 0.8 1.02 ¨« 0.9* 0.90 ¨« 0.8* 1.00 ¨« 0.7*Systolic 2.58 ¨« 0.28 1.03 ¨« 0.8* 0.97 ¨« 0.6* 1.03 ¨« 0.13*LV posterior wall (mm)Diastolic 1.50 ¨« 0.15 1.94 ¨« 0.17* 1.97 ¨« 0.13* 2.32 ¨« 0.22*?æSystolic 2.51 ¨« 0.27 2.56 ¨« 0.28 2.52 ¨« 0.16 2.88 ¨« 0.18*?æInfarct size (%) . 48.8 ¨« 5.6 51.3 ¨« 6.7 47.3 ¨« 7.5Sham, n = 11; HF-saline, n = 13; HF Ad-GFP, n = 14; HF Ad-b2-adrenoceptor, n = 13.*P < 0.01 vs. sham.?æP < 0.01 vs. HF saline and HF GFP.HF, heart failure; LV, left ventricle; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; ISO, isoprenaline.b2-adrenoceptor regulates angiogenesis in heart failure BJPBritish Journal of Pharmacology (2012) 166 2348.2361 2353Ad-b2-adrenoceptor-infected hearts compared with other HFgroups.Cardiac b2-adrenoceptors overexpression andVEGF/PKB/eNOS pathwayNext, we explored the effects of b2-adrenoceptor overexpressionon the cardiac VEGF/PKB/eNOS pathway, which isknown to have a critical role in the control of the angiogenicmechanisms in the heart (Lymperopoulos et al.,2008; 2010).At 2 weeks after gene transfer, all HF groups showedhigher cardiac phospho (p)-PKB/total (t)-PKB ratio comparedwith sham animals (Figure 5A). Noteworthy, in the HFb2-adrenoceptor group, PKB activation was associated withenhanced cardiac VEGF and p-eNOS/eNOS protein levelscompared with HF control groups. In contrast, HF-saline andHF-GFP groups did not show any increase in VEGF andp-eNOS protein expression, with levels comparable to thoseobserved in sham hearts. These results agree with previousdata, indicating that PKB activation is not always associatedwith enhanced activity of its downstream effectors, and, mostimportantly, that dissociation between PKB and eNOS/VEGFleads to negative cardiac remodelling (Shiojima et al., 2005;Shiojima and Walsh, 2006). Overall, these data stronglysuggest that cardiac b2-adrenoceptor overexpression is able torestore the integrity of the pro-angiogenic pathway and topromote coordinated cardiac and vascular growth in thepost-MI failing myocardium.Interestingly, 4 weeks post-gene therapy, the activation ofthe VEGF/PKB/eNOS pathway was blunted in the HF-b2-adrenoceptor group, whereas, in the HF control groups thep-PKB/t-PKB ratio was still higher compared with both HF-theb2-adrenoceptFigure 3(A) Representative LV cross sections and (B) echocardiographic M-mode recordings from of all study groups at the end of the study period. (C)LV internal diameter at diastole (LVIDd) (left), LVID at systole (LVIDs) (middle) and fractional shortening (FS) (right) as measured by echocardiography4 weeks after gene delivery. Sham, n = 11; HF-saline, n = 14; HF Ad-GFP, n = 11; HF Ad-b2-adrenoceptor (AR), n = 12. Bar = 10 mm. ANOVAanalysis and Bonferroni test among all groups. All data are expressed as mean ¨« SEM. *P < 0.05 vs. sham at basal or at each respective dose ofisoprenaline; ?æP < 0.05 vs. HF-saline and HF Ad-GFP at basal or at each respective dose of isoprenaline.BJP G Rengo et al.2354 British Journal of Pharmacology (2012) 166 2348.2361PKB activation was not associated with enhanced VEGF andeNOS protein levels.In order to link directly the effects of b2-adrenoceptoroverexpression to the activation of the VEGF/PKB/eNOSpathway, an additional group of Ad.b2-adrenoceptor HFinfected rats was treated with the specific b2-adrenoceptorantagonist ICI118 551 (0.2 mg?Ekg-1day-1) beginning 5 daysbefore gene delivery. As illustrated in Figure S2, ICI118551was able to completely prevent b2-adrenoceptoroverexpression-dependent activation of the pro-angiogenicsignalling in the hearts. This result suggests a direct effect ofb2-adrenoceptors on the activation of VEGF/PKB signalling.Interestingly, in b2-adrenoceptor-infected hearts, theVEGF/PKB/eNOS pathway, evaluated separately in cardiomyocytesand endothelial cells, was activated to a similarextent in both cell subtypes at 2 weeks after gene delivery (seesupplemental Figure S3). This indicates that the overall effecton angiogenesis activation in b2-adrenoceptor-overexpressinghearts could be mediated by pro-angiogenic stimuli originatingfrom both myocytes and EC, thus, confirming previousobservations suggesting the existence of reciprocal cross-talkmechanisms between the vasculature and cardiac myocytesthat regulate coronary angiogenesis and contractile function(Shiojima et al., 2005; Shiojima and Walsh, 2006).Impairment of cardiac angiogenesis and LVcontractility in post-ischaemic HFb2-adrenoceptor-/- miceTo further support the notion of a crucial role for theb2-adrenoceptor in the regulation of cardiac function andangiogenesis during HF, homozygous b2-adrenoceptor-/-(b2KO) or b2-adrenoceptor+/+ male mice littermates (controlgroup) underwent MI (to induce HF) or sham operation(Figure 6). One month after surgically induced MI, the mortalityrate was threefold higher in b2-adrenoceptor-/-compared with b2-adrenoceptor+/+ mice (Figure 6A).Echocardiography performed at this time point showedreduced FS in HF b2-adrenoceptor+/+ compared with shammice. The lack of b2-adrenoceptors was associated withfurther decrease in FS compared with HF b2-adrenoceptor+/+mice (Figure 6B and Supplemental Table 2).Interestingly, cardiac capillary density was significantlydecreased in sham b2-adrenoceptor-/- compared with shamb2-adrenoceptor+/+. Moreover, 1 month post-MI, both HFShamoperated0306090120Arterioles (mm mm.3)Fold increase in conductance15017000.20.40.6Capillaries ??m.2mL?Emin.1?E100 g.10.811.20200400600800100012001400160001234567CEA BCapillariesHF Ad-??2AR HF-GFP HF saline ShamArteriolesDBorderRemoteBorderRemoteBasalMax dilationHFsalineHFAd-GFPHFAD-??2ARShamoperatedHFsalineHFAd-GFPHFAD-??2ARShamoperatedHFsalineHFAd-GFPHFAD-??2ARShamoperatedHFsalineHFAd-GFPHFAD-??2AR*?æ*?æ*?æ*?æ*?æ*?æ* ******* ****Figure 4(A) Representative images of (left) Lectin Bandeiraea simplicifolia I staining of capillaries in LV sections and (right) of arterioles stained withantibodies against smooth muscle a-actin obtained from all study groups at 4 weeks post-gene therapy in the lateral wall far from the infarctedarea (remote). Magnification 40. Scale bar: 50 mm. (B) Histograms show data on capillary counts, and (C) arteriolar length density in either LVborder anterior and lateral, and remote zones in all study groups at 4 weeks after gene therapy (n = 5 for each group). (D) Average of myocardialblood flow at basal condition and after maximal coronary dilation by dipyridamole and of coronary reserve measured in all study groups at theend of the study period (n = 8 rats for each group). ANOVA analysis and Bonferroni test among all groups. All data are expressed as mean ¨« SEM.*P < 0.05 vs. sham; ?æP < 0.05 vs. HF-saline and HF Ad-GFP.b2-adrenoceptor regulates angiogenesis in heart failure BJPBritish Journal of Pharmacology (2012) 166 2348.2361 2355mouse lines showed a decrease in capillary density comparedwith the respective sham groups. However, HFb2-adrenoceptor-/- mice had significantly lower capillarydensity compared with HF b2-adrenoceptor+/+ (Figure 6C). Atthe molecular level, we investigated the VEGF/PKB/eNOSpathway by Western blotting in both b2-adrenoceptor-/- andb2-adrenoceptor+/+ mice 1 month post-sham operation orsurgically induced MI (Figure 6D). In HF b2-adrenoceptor+/+mice, we found an enhanced p-PKB/t-PKB ratio comparedwith both sham mouse lines. Interesti
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