Emmanuel Messas

Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study

BACKGROUND Heart failure develops after myocardial infarction and is a major cause of morbidity and mortality. The ability to direct differentiation of embryonic stem cells (ESC) towards a cardiomyogenic phenotype makes them an attractive therapeutic option for cardiac repair, but species-specific and individual-specific immunological imprinting remains a hurdle. Our aim was to ascertain whether the purported immune privilege of ESC allows for their cross-species engraftment in a clinically relevant large-animal model. METHODS We studied engraftment and differentiation of cardiac-committed mouse ESC in 18 sheep in which a myocardial infarction had been induced; nine controls received medium and nine sheep (five of which were immunosuppressed) received ESC. The gain in myocardial function was measured by echocardiography 1 month after cell transplantation. FINDINGS Cardiac-committed murine ESC engrafted in infarcted myocardium of immunosuppressed and immunocompetent sheep, and differentiated into mature cardiomyocytes that expressed connexins. Colonisation of the scar area by ESC was accompanied by a functional benefit of the damaged myocardium. Left-ventricular ejection fraction deteriorated in the control group by a median of 9.9% (range -20 to 0.3) relative to baseline (p=0.011) whereas in the treated group it improved by 6.6% (-5.7 to 50.8; comparison between groups p=0.002). INTERPRETATION These findings obtained in a clinically relevant large-animal model of heart failure strengthen the potential therapeutic use of ESC to regenerate the severely dysfunctional myocardium and bring additional evidence for an immune privilege of these cells.

Skeletal myoblast transplantation in ischemic heart failure: long-term follow-up of the first phase I cohort of patients.

Background— Skeletal myoblast (SM) transplantation (Tx) in a post-myocardial infarction (MI) scar experimentally improves left ventricular (LV) ejection fraction (EF). Short-term follow-up (FU) studies have suggested that a similar benefit could clinically occur despite an increased risk of LV arrhythmias. Methods and Results— We report the long-term FU of the first worldwide cohort of grafted patients (n =9, 61.8±11.6 years, previous MI, EF ≤35%) operated on (autologous SM Tx and bypass surgery) in 2000 to 2001 and evaluated before Tx, at 1 month (M1) and at a median FU of 52 (18 to 58) months after Tx (37 patient-years). NYHA class improved from 2.5±0.5 to 1.8±0.4 at M1 (P=0.004 versus baseline) and 1.7±0.5 at FU (P=not significant versus M1; P=0.0007 versus baseline). EF increased from 24.3±4% to 31±4.1% at M1 (+28%, P=0.001 versus baseline) and remained stable thereafter (28.7±8.1%, +18% versus baseline). There were 5 hospitalizations for heart failure in 3 patients at 28.6±9.9 months, allowing implant in 2 patients with a resynchronization pacemaker. An automatic cardiac defibrillator (ACD) was implanted in 5 patients for nonsustained (n =1) or sustained (n =4) ventricular tachycardia at 12.2±18.6 (1 to 45) months. Despite a beta-blocker/amiodarone combination therapy, there were 14 appropriate shocks for 3 arrhythmic storms in 3 patients at 6, 7, and 18 months after ACD implantation. Conclusions— In this cohort of severe heart failure patients both clinical status and EF stably improve over time with a strikingly low incidence of hospitalizations for heart failure (0.13/patient-years) and the arrhythmic risk can be controlled by medical therapy and/or on-request ACD implantation.

Factors affecting functional outcome after autologous skeletal myoblast transplantation

BACKGROUND This study assessed the extent to which the initial degree of functional impairment and the number of injected cells may influence the functional improvement provided by autologous skeletal myoblast transplantation into infarcted myocardium. METHODS One week after left coronary artery ligation, 44 rats received into the infarcted scar, autologous skeletal myoblasts expanded in vitro for 7 days (mean, 3.5 x 10(6), n = 21), or culture medium alone (controls, n = 23). Left ventricular function was assessed by two-dimensional echocardiography. RESULTS When transplanted hearts were stratified according to their baseline ejection fraction, a significant improvement occurred at 2 months in the less than 25% (from 21.4% to 37%), 25% to 35% (from 29% to 43.8%), and in the 35% to 40% (from 37.2% to 41.7%) groups, compared to controls (p = 0.048, 0.0057, and 0.034, respectively), but not in the more than 40% stratum. A significant linear relationship was found between the improvement in ejection fraction and the number of injected myoblasts, both at 1 and 2 months after transplantation (p < 0.0001). CONCLUSIONS Autologous myoblast transplantation is functionally effective over a wide range of postinfarct ejection fractions, including in the sickest hearts provided that they are injected with a sufficiently high number of cells.

Quantitative Assessment of Arterial Wall Biomechanical Properties Using Shear Wave Imaging

A new ultrasound-based technique is proposed to assess the arterial stiffness: the radiation force of an ultrasonic beam focused on the arterial wall induces a transient shear wave (∼10 ms) whose propagation is tracked by ultrafast imaging. The large and high-frequency content (100 to 1500 Hz) of the induced wave enables studying the wave dispersion, which is shown experimentally in vitro and numerically to be linked to arterial wall stiffness and geometry. The proposed method is applied in vivo. By repeating the acquisition up to 10 times per second (theoretical maximal frame rate is ∼100 Hz), it is possible to assess in vivo the arterial wall elasticity dynamics: shear modulus of a healthy volunteer carotid wall is shown to vary strongly during the cardiac cycle and measured to be 130 ± 15 kPa in systole and 80 ± 10 kPa in diastole.

Differentiation In Vivo of Cardiac Committed Human Embryonic Stem Cells in Postmyocardial Infarcted Rats

Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES‐1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein‐2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real‐time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and α‐actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in‐scar injections of either culture medium (controls) or BMP2 (±SU5402)‐treated HES cells. After 2 months, human cells were detected by anti‐human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription‐PCR and immunofluorescence using an anti‐β myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac‐specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Interleukin-1 Receptor Signaling Mediates Atherosclerosis Associated With Bacterial Exposure and/or a High-Fat Diet in a Murine Apolipoprotein E Heterozygote Model Pharmacotherapeutic Implications

Background—Current data demonstrate that progressive atherosclerosis is associated with activation of the inflammatory process, as evidenced by systemic elevations of molecules such as tumor necrosis factor, interleukin (IL)-6, and IL-1. It has been postulated that inflammatory events within an atherogenic lesion are induced by oxidized LDL. Recent evidence suggests that infectious agents, including those that cause periodontal disease, may also play an important role. Studies presented here tested the hypothesis that IL-1 receptor (IL-1R1) signaling plays a crucial role in bacteria- and/or high-fat diet (HFD)–enhanced atherogenesis. Methods and Results—Ten-week-old ApoE+/− mice lacking either 1 IL-1R1 allele (ApoE+/−/IL-1R1+/−) or 2 IL-1R1 alleles (ApoE+/−/IL-1R1−/−) fed either an HFD or regular chow were inoculated intravenously with live Porphyromonas gingivalis (P gingivalis) (107 CFU), an important periodontal pathogen, or vehicle once per week for 14 or 24 consecutive weeks. Histomorphometry of plaque cross-sectional area in the proximal aortas, en face measurement of plaque area over the aortic trees, and ELISA for systemic proinflammatory mediators were performed. Atherosclerotic lesions of proximal aortas and aortic tree were substantially reduced in ApoE+/−/IL-1R1−/− mice than in ApoE+/−/IL-1R1+/− mice challenged with P gingivalis. At 24 weeks after P gingivalis inoculation, proximal aortic lesion size quantified by histomorphometry was 5-fold–reduced in chow-fed ApoE+/−/IL-1R1−/− mice than in ApoE+/−/IL-1R1+/− mice (P<0.05). In the HFD group, ApoE+/−/IL-1R1−/− mice exhibited marked attenuation of the progression of atherosclerotic lesions (78% to 97%), with and without P gingivalis inoculation (P<0.05) Conclusion—Ablation of IL-1R1 under P gingivalis challenge and/or an HFD reduced the progression of atherosclerotic plaques. These results indicate that IL-1 plays a crucial role in bacteria- and/or HFD-enhanced atherogenesis.

Efficacy of Chordal Cutting to Relieve Chronic Persistent Ischemic Mitral Regurgitation

Background—Mitral regurgitation (MR) conveys adverse prognosis in ischemic heart disease. Leaflet closure is restricted by tethering to displaced papillary muscles, and is, therefore, incompletely treated by annular reduction. In an acute ischemic model, we reduced such MR by cutting a limited number of critically positioned chordae to the leaflet base that most restrict closure but are not required to prevent prolapse. Whether this is effective without prolapse, recurrent MR, or left ventricular (LV) failure in chronic persistent ischemic MR, despite greater LV remodeling, remains to be established. Therefore, we studied 7 sheep with chronic inferobasal infarcts known to produce progressive MR over 2 months. In all of those sheep, after a mean of 4.1 months, the 2 central basal (intermediate) chordae were cut at the chronic ischemic MR stage. 3-Dimensional echo quantified MR, LV function, and valve geometry. Five other sheep were followed for a mean of 7.8±1.2 months after inferobasal infarction with chordal cutting. Results—All 7 of the sheep with chronic ischemic MR (increased from 1.4±0.4 to 11.1±0.5 mL/beat, regurgitant fraction=39.0±4.2%, P <0.0001) showed anterior leaflet angulation at the basal chord insertion. Although end-systolic volume had doubled, cutting the 2 central basal chordae significantly decreased the MR to baseline (P <0.0001) without prolapse or decline in EF (41.1±1.5% to 42.6±1.6%, P =not significant [NS]). The five sheep with long-term follow-up showed no prolapse or MR, and no significant post-infarct decrease in LV ejection fraction (EF; 38.9±2.4% to 41.4±1.2%, P =NS). Conclusion—Cutting a minimum number of basal (intermediate) chordae can improve coaptation and reduce chronic persistent ischemic MR without impairing LVEF. No adverse effects were noted long-term after chordal cutting at the time of infarction.

Transplantation of Autologous Fresh Bone Marrow Into Infarcted Myocardium: A Word of Caution

Background—As the benefits of extemporaneous transplantation (Tx) of fresh (unfractionated) autologous bone marrow (BM) have been primarily studied in the setting of acute myocardial infarction, we assessed whether this approach could be effective for regenerating chronically infarcted myocardium. Methods and Results—Myocardial infarction was created in 18 sheep by ligation of circumflex arterial branches. Three weeks later, BM was aspirated from the iliac crest, washed, labeled with the fluorescent dye Dil and reinjected (mean: 422×106 cells in 3 mL) in 10 sites across the infarcted area through the reopened thoracotomy (n=9). Nine controls received culture medium. Left ventricular (LV) function was assessed before and 2 months after Tx by two-dimensional echocardiography whereas transmural velocity gradients were measured using M-mode tissue Doppler imaging at the center of the infarcted/grafted area. Formalin-fixed hearts were processed for the detection of grafted cells and angiogenesis. LV ejection fraction deteriorated similarly in the Tx and control groups (from 42±5% to 30±4% and from 40±4% to 31±1%, respectively; P =0.86). Likewise, BM Tx failed to prevent LV dilatation and impairment of the global wall motion score. The decrease in regional systolic velocity gradients (s−1) featured a similar pattern (Tx group: from 0.77±0.11 to 0.31±0.07; control group: from 0.73±0.10 to 0.50±0.07; P =0.06). Histologically, there was neither BM tissue engraftment, except for a few scattered Dil-positive macrophages in the infarcted fibrotic areas nor transdifferentiation of BM cells into endothelial cells. Conclusion—These data caution against the functional efficacy of extemporaneous Tx of fresh unfractionated BM into postinfarction scars.

In Vivo Quantitative Mapping of Myocardial Stiffening and Transmural Anisotropy During the Cardiac Cycle

Shear wave imaging was evaluated for the in vivo assessment of myocardial biomechanical properties on ten open chest sheep. The use of dedicated ultrasonic sequences implemented on a very high frame rate ultrasonic scanner (>; 5000 frames per second) enables the estimation of the quantitative shear modulus of myocardium several times during one cardiac cycle. A 128 element probe remotely generates a shear wave thanks to the radiation force induced by a focused ultrasonic burst. The resulting shear wave propagation is tracked using the same probe by cross-correlating successive ultrasonic images acquired at a very high frame rate. The shear wave speed estimated at each location in the ultrasonic image gives access to the local myocardial stiffness (shear modulus μ). The technique was found to be reproducible (standard deviation <; 3%) and able to estimate both systolic and diastolic stiffness on each sheep (respectively μdias ≈ 2 kPa and μsyst ≈ 30 kPa). Moreover, the ability of the proposed method to polarize the shear wave generation and propagation along a chosen axis permits the study the local elastic anisotropy of myocardial muscle. As expected, myocardial elastic anisotropy is found to vary with muscle depth. The real time capabilities and potential of Shear Wave Imaging using ultrafast scanners for cardiac applications is finally illustrated by studying the dynamics of this fractional anisotropy during the cardiac cycle.

Mapping Myocardial Fiber Orientation Using Echocardiography-Based Shear Wave Imaging

The assessment of disrupted myocardial fiber arrangement may help to understand and diagnose hypertrophic or ischemic cardiomyopathy. We hereby proposed and developed shear wave imaging (SWI), which is an echocardiography-based, noninvasive, real-time, and easy-to-use technique, to map myofiber orientation. Five in vitro porcine and three in vivo open-chest ovine hearts were studied. Known in physics, shear wave propagates faster along than across the fiber direction. SWI is a technique that can generate shear waves travelling in different directions with respect to each myocardial layer. SWI further analyzed the shear wave velocity across the entire left-ventricular (LV) myocardial thickness, ranging between 10 (diastole) and 25 mm (systole), with a resolution of 0.2 mm in the middle segment of the LV anterior wall region. The fiber angle at each myocardial layer was thus estimated by finding the maximum shear wave speed. In the in vitro porcine myocardium (n = 5), the SWI-estimated fiber angles gradually changed from +800 ± 7° (endocardium) to +30° ± 13° (midwall) and -40° ± 10° (epicardium) with 0° aligning with the circumference of the heart. This transmural fiber orientation was well correlated with histology findings (r2 - 0.91 ± 0.02, p <; 0.0001). SWI further succeeded in mapping the transmural fiber orientation in three beating ovine hearts in vivo. At midsystole, the average fiber orientation exhibited 71° ± 13° (endocardium), 27° ± 8° (midwall), and - 26° ± 30° (epicardium). We demonstrated the capability of SWI in mapping myocardial fiber orientation in vitro and in vivo. SWI may serve as a new tool for the noninvasive characterization of myocardial fiber structure.