Validating intravascular imaging with serial optical coherence tomography and confocal fluorescence microscopy

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology

Altération du système bêta-adrénertique cardiaque au cours de la cardiotoxicité induite par la doxorubicine

NANTES-BU Sciences (441092104) / SudocSudocFranceF

New Insights in Research About Acute Ischemic Myocardial Injury and Inflammation

International audienceRecognition that inflammation may contribute to the pathogenesis of various cardiac diseases has naturally led to the evaluation of the therapeutic potential of a range of anti-inflammatory approaches. Unfortunately, results in most settings have been disappointing. The majority of novel approaches fail despite promising preclinical data, partly attributable to off-target effects. The purpose of this review, focused on inflammation following acute myocardial ischemia, is to give a brief overview of the new insights regarding research on pro-inflammatory signaling cascades that could be targeted for cardioprotective therapeutic developments

Increased beta2-adrenoceptors in doxorubicin-induced cardiomyopathy in rat.

BACKGROUND: The toxicity of doxorubicin, leading to an irreversible heart failure, limits its use as chemotherapeutic agent. The beneficial effects of early administration of β-blocker were reported in patients with heart failure due to doxorubicin, suggesting an important role of β-adrenoceptors (β-ARs). This study aimed to identify a putative target (β-AR and/or its effectors) at the early phase of a chronic doxorubicin-induced cardiomyopathy (Dox-CM) in a rat model. METHODOLOGY: Dox-CM was induced by six doxorubicin injections (cumulative dose: 15 mg x kg(-1)) and validated by echocardiography and left ventricle (LV) catheterization. The β-AR protein expressions in LV were evaluated by western-blot at days 35 (d35) and 70 (d70) after the first doxorubicin injection. Ex vivo cardiac contractility (dP/dtmax, dP/dtmin) was evaluated on isolated heart in response to specific β-AR stimulations at d35. RESULTS: At d35, Dox-CM hearts were characterized by mild LV systolic and diastolic dysfunctions, which were exacerbated at d70. In Dox-CM hearts, β3-AR expression was only decreased at d70 (-37±8%). At d35, β1-AR expression was decreased by 68±6%, but ex vivo β1-AR function was preserved due to, at least in part, an increased adenylyl cyclase response assessed by forskolin. β2-AR expression was increased both at d35 (+58±22%) and d70 (+174±35%), with an increase of ex vivo β2-AR response at d35. Inhibition of Gi protein with pertussis toxin did not affect β2-AR response in Dox-CM hearts, suggesting a decoupling of β2-AR to Gi protein. CONCLUSION: This study highlights the β1/β2-AR imbalance in early Dox-CM and reveals the important role that β2-AR/Gi coupling could play in this pathology. Our results suggest that β2-AR could be an interesting target at early stage of Dox-CM

252 Cardiac β3-adrenoceptors as a new therapeutic target in dilated cardiomyopathy

Introductionβ-adrenergic system is altered in heart failure (HF) due to non-ischemic dilated cardiomyopathy (DCM). There are few data concerning the relative contribution of β1- and β3-adrenoceptor subtypes (β1- and β3-AR) during DCM development. We evaluated the expression and the role of each β-AR subtype in this pathology.MethodsDCM rat model is performed by doxorubicin injections (cumulative dose: 15 mg.kg-1) and validated by in vivo measurements with echocardiography-doppler. The variations of β1- and β3-AR transcript expression in left ventricle (LV) are evaluated by real-time RT-PCR. The ex vivo cardiac responses induced by selective β3-AR or non-selective β-AR stimulations are evaluated on isolated perfused heart.ResultsDCM rats present LV dilation, systolic and diastolic dysfunction (see table). Compared to controls, β1-AR transcripts and β3-AR transcripts are increased in DCM LV (+36%, n=8, p<0.05 and +358%, n=8, p<0.05). Ex vivo parameters are summarized in the table.Table: Basal parameters and maximum values obtained by non-selective β1-AR stimulation (isoproterenol) or selective β3-AR stimulation (SR58611A). Results are expressed by mean ± SEM.*: p<0.001 vs Control, £: p<0.001 vs Basal.Control rats (n=9-31)DCM rats (n=10-31)Basal in vivoLV end-diastolic diameter (mm)8.82±0.337.61±0.17 *LV ejection fraction (%)83.0±1.971.2±2.8*LV Isovolumic relaxation time (ms)21.41±1.1332.39±1.10*Basal ex vivoDP/dt max (mmHg.s-1)2035±3652669±504*DP/dt min (mmHg.s-1)–1258±226–1847±349*Isoproterenol ex vivo (1 μM)DP/dt max (mmHg.s-1)5263±17544373±1383*DP/dt min (mmHg.s-1)–3815±1271–3227±1020*SR58611A ex vivo (1 μM)DP/dt max (mmHg.s-1)1722±4971930±611£DP/dt min (mmHg.s-1)–1027±297–1138±360£ConclusionDCM induces a β3-AR gene over-expression, associated to an increase of β3AR-induced negative inotropic and lusitropic effects. Those results could partly explain the alteration of isoproterenol response in our model, suggesting that β3-AR could be a new therapeutic target in DCM

Cardiac inflammation and diastolic dysfunction in hypercholesterolemic rabbits.

BackgroundLeft ventricular diastolic dysfunction (LVDD) is present in more than 50% of patients suffering from heart failure. LVDD animal models are limited and its underlying mechanisms remain largely unknown. Aortic valve stenosis (AVS) may cause LVDD, and we recently reported LVDD in an AVS rabbit model. Here we aimed to develop a rabbit model of LVDD without AVS.MethodsRabbits were fed with a 0.5% cholesterol-enriched diet (n = 9) or normal diet (n = 8) until they developed LVDD defined by a value of the echocardiographic parameter E/Em ratio higher than the mean at baseline + 2SD. Rabbits were then fed a 0.2% cholesterol-enriched diet for 4 weeks (average total diet duration: 20 weeks). Detailed cardiac structure and function measurements were assessed by echocardiography at baseline, weeks 8, 12 and 14 to 20, when applicable. Histological analyses and RT-qPCR were performed on LV samples.ResultsThe hypercholesterolemic diet induced LVDD without systolic dysfunction or AVS, as shown by multiple echocardiographic parameters, including early filling mitral peak velocity and deceleration rate, Em/Am ratio and E/Em ratio (all pConclusionRabbits fed with a cholesterol-enriched diet develop LVDD with preserved systolic function and evidence of cardiac inflammation and oxidative stress. This rabbit model may be used in future studies to test treatment strategies against LVDD

Increased β2-adrenergic vasorelaxation at the early phase of endotoxemic shock in rats

International audienceBACKGROUND AND PURPOSE: The early management of the cardiovascular dysfunction of septic shock is critical as it is associated with a poor outcome. Although the use of catecholamines is a common therapy in this syndrome, no data are available on the involvement of β-adrenoceptor (β-AR) subtypes and only few studies report an alteration of β-adrenergic-induced vasodilation in septic shock. The purpose of the study was to evaluate vascular β1, β2 and β3-AR expression and function in an endotoxemic rat model. EXPERIMENTAL APPROACH: Endotoxemia was induced in rats by intravenous injection of lipopolysaccharide (LPS). β1, β2 and β3-AR mRNA expression was evaluated by RT-PCR in aorta and vascular β1, β2 and β3-AR responses were determined on conducting (aorta) and/or resistance (mesenteric and renal) arteries by constructing relaxation curves in response to different β-AR agonists. RESULTS: The maximal effect of isoproterenol decreased by 31 to 61% in the three vascular beds of LPS-treated rats compared to controls. In aortas from LPS-treated rats, β1 and β3-AR mRNA expression was decreased and associated to a reduced β1 and β3-induced vasodilation. Conversely, albeit β2-AR mRNA was unchanged, the maximal β2-AR-induced vasodilation increased by 49% in aortas from LPS-treated rats compared to controls. This increase was not affected by endothelium removal but was abolished in the presence of a β2-AR antagonist or an adenylate cyclase inhibitor. CONCLUSIONS: In endotoxemia, β2-AR vasodilation was increased by a potential recruitment of β2-AR located on smooth muscle cells. This study suggests that vascular β2-AR should be a putative new therapeutic target in septic shock

β₃-AR expression and function in Dox-CM hearts.

<p>A. Representative β₃-AR immunoblotting at days 35 (d35) and 70 (d70). B. β₃-AR protein quantification at day 35. C. β₃-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dt_max), E. lusitropic (dP/dt_min) and F. chronotropic (HR) effects of increasing concentrations of SR 58611A (10⁻⁹ to 10⁻⁵ M) were evaluated on isolated perfused hearts. **: <i>P</i><0.001 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

Gi protein expression and involvement in β₂-AR cardiac contractility.

<p><b>A.</b> Representative Giα₂ immunoblotting at days 35 (d35) and 70 (d70). <b>B.</b> Giα₂ protein quantification at day 35. <b>C.</b> Giα₂ protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. <b>D.</b> Cardiac inotropic (dP/dt_max), <b>E.</b> lusitropic (dP/dt_min) and <b>F.</b> chronotropic (HR) effects of increasing concentrations of isoproterenol (10⁻⁹ to 10⁻⁴ M) in the presence of 10⁻⁶ M of CGP-20712A and 10⁻⁶ M of L-748,337 were evaluated on isolated perfused hearts pre-treated or not with 4 µg.L⁻¹ of pertussis toxin (PTX) a Gi protein inhibitor. B: Baseline; B+A; Baseline in the presence of antagonists. *: <i>P</i><0.05 <i>vs</i> Ctrl; **: <i>P</i><0.001 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

Gsα protein expression and forskolin response.

<p><b>A.</b> Representative Gsα immunoblotting at days 35 (d35) and 70 (d70). <b>B.</b> Gsα protein quantification at day 35. <b>C.</b> Gsα protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. <b>D.</b> Cardiac inotropic (dP/dt_max), <b>E.</b> lusitropic (dP/dt_min) and <b>F.</b> chronotropic (HR) effects of increasing concentrations of the adenylyl cyclase activator forskolin (3.10⁻⁸ to 10⁻⁵ M) were evaluated on isolated perfused hearts. *: <i>P</i><0.05 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p