Irene C. Turnbull

Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium

Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs, >90% troponin‐positive) were mixed with collagen and cultured on force‐sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18±0.48 Hz). Microstructural features and mRNA expression of cardiac‐specific genes (α‐MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank‐Starling mechanism, generating an average maximum twitch stress of 660 μN/mm2 at Lmax, approaching values in newborn human myocardium. Dose‐response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 μM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3‐D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.—Turnbull, I. C., Karakikes, I., Serrao, G. W., Backeris, P., Lee, J.‐J., Xie, C., Senyei, G., Gordon, R. E., Li, R. A., Akar, F. G., Hajjar, R. J., Hulot, J.‐S., Costa, K. D. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J. 28, 644–654 (2014). www.fasebj.org

Therapeutic Efficacy of AAV1.SERCA2a in Monocrotaline-Induced Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is characterized by dysregulated proliferation of pulmonary artery smooth muscle cells leading to (mal)adaptive vascular remodeling. In the systemic circulation, vascular injury is associated with downregulation of sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and alterations in Ca2+ homeostasis in vascular smooth muscle cells that stimulate proliferation. We, therefore, hypothesized that downregulation of SERCA2a is permissive for pulmonary vascular remodeling and the development of PAH. Methods and Results— SERCA2a expression was decreased significantly in remodeled pulmonary arteries from patients with PAH and the rat monocrotaline model of PAH in comparison with controls. In human pulmonary artery smooth muscle cells in vitro, SERCA2a overexpression by gene transfer decreased proliferation and migration significantly by inhibiting NFAT/STAT3. Overexpresion of SERCA2a in human pulmonary artery endothelial cells in vitro increased endothelial nitric oxide synthase expression and activation. In monocrotaline rats with established PAH, gene transfer of SERCA2a via intratracheal delivery of aerosolized adeno-associated virus serotype 1 (AAV1) carrying the human SERCA2a gene (AAV1.SERCA2a) decreased pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and fibrosis in comparison with monocrotaline-PAH rats treated with a control AAV1 carrying &bgr;-galactosidase or saline. In a prevention protocol, aerosolized AAV1.SERCA2a delivered at the time of monocrotaline administration limited adverse hemodynamic profiles and indices of pulmonary and cardiac remodeling in comparison with rats administered AAV1 carrying &bgr;-galactosidase or saline. Conclusions— Downregulation of SERCA2a plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PAH. Selective pulmonary SERCA2a gene transfer may offer benefit as a therapeutic intervention in PAH.

Improved outcomes are associated with multilevel endovascular intervention involving the tibial vessels compared with isolated tibial intervention

OBJECTIVE Endovascular intervention is increasingly accepted as an alternative to surgery for the treatment of tibial vessel disease. Tibial vessel disease can occur in isolation or in conjunction with disease that involves the proximal lower extremity vasculature (multilevel disease). This study evaluated the overall efficacy of endovascular intervention for tibial vessel disease and whether the requirement for single-level compared with multilevel intervention affected outcomes. METHODS This study evaluated a consecutive unselected group of patients who underwent an infrapopliteal intervention from November 2002 to February 2008. The primary end points evaluated were technical success, limb salvage, primary patency, and secondary patency. The secondary end points evaluated were 30-day access site (ie, hematoma, pseudoaneurysm, and wound infection), intervention site (ie, thrombosis), and systemic (ie, acute renal failure, myocardial infarction, and mortality) complications. Patency and limb salvage were evaluated using Kaplan-Meier life-table analyses and compared using Cox regression analysis. P < .05 was considered statistically significant. RESULTS The study comprised 85 patients, 89 limbs, and 114 procedures. Age was 72.4 +/- 13.1 years, 67% were men, and follow-up was 245.8 +/- 290.8 days. The technical success rate for all procedures was 91%. Limb salvage rates for patients with critical limb ischemia at 6, 12 and 18 months were 85% +/- 0%, 81% +/- 0%, and 69% +/- 0%, respectively. For the complete patient cohort, primary patency rates at 6, 12 and 18 months were 68% +/- 6%, 50% +/- 8%, and 37% +/- 9%, respectively, and secondary patency rates were 81% +/- 5%, 71% +/- 7%, and 63% +/- 8%. Multilevel intervention was associated with significantly improved secondary patency compared with single-level intervention (P = .045). CONCLUSIONS Patency and limb salvage rates for endovascular treatment of tibial vessel disease in this study are comparable with prior reports and with historical surgical controls. Patients who undergo multilevel intervention involving the tibial vessels exhibit improved secondary patency compared with those who undergo intervention for lesions isolated to the tibial vessels. This may reflect increased distal disease burden for patients who undergo isolated tibial intervention. The study data suggest that the presence of multilevel disease should not preclude an attempt at percutaneous revascularization. Further study is required before formulating definitive recommendations for the endovascular treatment of tibial vessel disease.

Delivery of gelfoam-enabled cells and vectors into the pericardial space using a percutaneous approach in a porcine model

Intrapericardial drug delivery is a promising procedure, with the ability to localize therapeutics with the heart. Gelfoam particles are nontoxic, inexpensive, nonimmunogenic and biodegradable compounds that can be used to deliver therapeutic agents. We developed a new percutaneous approach method for intrapericardial injection, puncturing the pericardial sac safely under fluoroscopy and intravascular ultrasound (IVUS) guidance. In a porcine model of myocardial infarction (MI), we deployed gelfoam particles carrying either (a) autologous mesenchymal stem cells (MSCs) or (b) an adenovirus encoding enhanced green fluorescent protein (eGFP) 48 h post-MI. The presence of MSCs and viral infection at the infarct zone was confirmed by immunoflourescence and PCR. Puncture was performed successfully in 16 animals. Using IVUS, we successfully determined the size of the pericardial space before the puncture, and safely accessed that space in setting of pericardial effusion and also adhesions induced by the MI. Intrapericardial injection of gelfoam was safe and reliable. Presence of the MSCs and eGFP expression from adenovirus in the myocardium were confirmed after delivery. Our novel percutaneous approach to deliver (stem-) cells or adenovirus was safe and efficient in this pre-clinical model. IVUS-guided delivery is a minimally invasive procedure that seems to be a promising new strategy to deliver therapeutic agents locally to the heart.

Five-year results for the Talent enhanced Low Profile System abdominal stent graft pivotal trial including early and long-term safety and efficacy

OBJECTIVES The pivotal trial of the Talent enhanced Low Profile System (eLPS; Medtronic Vascular, Santa Rosa, Calif) stent graft evaluated short and long-term safety and efficacy of endovascular aneurysm repair (EVAR). These data and a confirmatory group assessing the performance of the CoilTrac delivery system supported the United States premarket approval application for the device. METHODS The pivotal trial was a prospective, nonrandomized study conducted at 13 sites from February 2002 to April 2003. The study group (n = 166) underwent EVAR using the Talent eLPS stent graft. The control group (n = 243) underwent open surgical AAA repair. Data for this group were obtained from the Society for Vascular Surgery Endovascular AAA Surgical Controls project. Outcomes were compared at 30 days and 12 months. Additional 5-year follow-up was obtained for the eLPS group. A single-center cohort of 137 patients was the confirmatory group for the assessment of the clinical performance of the CoilTrac delivery system, with analysis of outcomes <or=30 days from the procedure. RESULTS AAA anatomy with neck length as short as 3 mm and maximum neck diameter of 32 mm were included in the eLPS group. EVAR was superior to open repair for periprocedural outcomes, including mean procedure duration (167.3 vs 196.4 minutes, P < .001), blood transfusion (18.2% vs 56.8%, P < .001), median intensive care unit stay (19.3 vs 74.3 hours, P < .001), and mean hospital stay (3.6 vs 8.2 days, P < .001). Freedom from major adverse events was 89.2% for EVAR at 30 days vs 44.0% (P < .001) and 81.3% vs 42.4% at 1 year (P < .001). Freedom from all-cause mortality and aneurysm-related mortality (ARM) was 93.7% and 98.2% for EVAR vs 92.4% and 96.7% for the controls. Through 5 years for the EVAR group, rates of freedom from all-cause mortality, ARM, aneurysm rupture, and conversion to surgery were 69.8%, 96.5%, 98.2%, and 99.1%, respectively, with one conversion to surgery, 25 secondary reinterventions, and five site-reported instances of stent graft migration. The technical success rate for the CoilTrac confirmatory group was 100%, with no aneurysm rupture or conversion to open repair at 30 days. The 30-day all-cause mortality rate was 1.5% (2 of 137). CONCLUSIONS In a population with challenging anatomic characteristics, EVAR with the Talent eLPS and use of the CoilTrac delivery system compared favorably with open repair through 1 year. Sustained protection from ARM, with minimal reinterventions, was attained through 5 years.

Outcome of elective endovascular abdominal aortic aneurysm repair in octogenarians and nonagenarians

OBJECTIVE Compared to open repair of abdominal aortic aneurysms (AAA), endovascular aneurysm repair (EVAR) is associated with decreased perioperative morbidity and mortality. This study sought to examine the outcomes of EVAR in patients >or=80 years of age. METHODS This was a retrospective review from a prospectively maintained computerized database. A total of 322 patients aged >or=80 underwent elective EVAR from January 1997 to November 2007. Mean age was 84 years +/- 3.4 years (range, 80-95 years), and 78.5% were male. Mean aneurysm size was 62 mm +/- 12 mm (range, 39-110 mm). RESULTS Mean procedural blood loss was 350 mL (range, 50-2700 mL), and 13.9% required intraoperative transfusion. Mean length of postoperative stay was 2.46 days (median, 1 day; range, 1-42 days), with 54.3% of patients discharged on the first postoperative day. There were 25 (7.8%) perioperative major adverse events. The most common were categorized as device-related (6), cardiac (4), gastrointestinal (4), and bleeding/hematoma (3). The perioperative 30-day mortality rate was 3.1% (10 of 322). Mean follow-up was 25.7 months (range, 1-110 months). Overall, 47 patients (14.6%) required secondary intervention, 7 patients (2.2%) underwent conversion to open repair, and 4 patients (1.2%) died from AAA rupture. Endoleaks occurred in 95 patients (29.4%), with 20 type I, 48 type II, and 27 of indeterminate type; of these, 10 patients with type I endoleaks underwent secondary intervention. Freedom from all-cause mortality at 1 year was 84.3% and at 5 years was 27.4%. Freedom from aneurysm-related mortality at 5 years was 92.9%. CONCLUSION EVAR in octogenarians is associated with high procedural success and low perioperative morbidity and mortality. The midterm results of this study support the use of EVAR in this patient population. Further studies are needed to predict short-term and long-term mortality risk, and treatment for other causes of death.

Aortic Implantation of Mesenchymal Stem Cells after Aneurysm Injury in a Porcine Model

BACKGROUND Cell-based therapies are being evaluated in the setting of degenerative pathophysiologic conditions. The search for the ideal method of delivery and improvement in cell engraftment continue to pose a challenge. This study explores the feasibility of introducing mesenchymal stem cells (MSC) following aortic injury in a porcine model. METHODS Bone marrow-derived MSC were obtained from eight pigs, characterized for the MSC markers CD13 and CD 29, labeled with green fluorescent protein (GFP), and collected for autologous injection in a porcine model of abdominal aortic aneurysm (AAA). The pigs were euthanized (1-7 d) after the procedure to assess the histologic characteristics and presence of MSC in the aortic tissue. Negative controls included noninjured aorta. Tracking of the MSC was conducted by the identification of the GFP-labeled cells using immunofluorescence. RESULTS AAA sections stained with hematoxylin and eosin showed disorganization of the aortic tissue; collagen-muscle-elastin stain demonstrated fragmentation of elastin fibers. The presence of the implanted MSC in the aortic wall was evidenced by fluorescent microscopy showing GFP labeled cells. Engraftment of MSC up to 7 d after introduction was observed. CONCLUSION Autologous implantation of bone marrow-derived MSC following aortic injury in a porcine model may be successfully accomplished. The long-term impact and therapeutic value of such cell-based therapy will require further investigation.

Safety and efficacy of carotid angioplasty and stenting for radiation-associated carotid artery stenosis

INTRODUCTION Prior neck irradiation may induce atherosclerosis in the carotid artery and is considered an indication for carotid angioplasty and stenting (CAS). This study sought to evaluate the effect of neck radiation therapy (XRT) on the rate of restenosis and embolic potential in patients undergoing CAS. METHODS Two hundred ten CAS procedures were performed on 193 patients (XRT [N = 28], non-XRT [N = 182]). Mean follow-up was 347 +/- 339 days (median, 305 days; range, 16-1354 days). Duplex velocity criteria for restenosis after CAS were: >50% restenosis (peak systolic velocity [PSV] > 125 cm/sec, end diastolic velocity [EDV] 40-99 cm/sec, and internal carotid artery to common carotid artery systolic ratio [ICA/CCA] > 2.0); >70% restenosis (PSV>230 cm/sec, EDV>100 cm/sec, and ICA/CCA ratio >4.0). Restenosis >70% was confirmed by digital subtraction angiography. Additional endpoints included groin hematoma, groin pseudoaneurysm, myocardial infarction, stroke, mortality, and the combined myocardial infarction/stroke/mortality rate. Captured particulate data was obtained from microporous filters used during CAS. Nineteen XRT and 128 non-XRT consecutive filters were analyzed. Photomicroscopy was performed along three axes for each filter, and the quantity and size of the captured particles were analyzed using video image analysis software. RESULTS There were more men (XRT: 85.7% vs. non-XRT: 52.8%, P < .001) and prior surgical neck dissections in the XRT patients (XRT: 82.1% vs. non-XRT: 4.7%, P < .001). Pre-procedural stenosis did not differ significantly betweeen the two groups (XRT: 86.5% +/- 8.9% [range, 70%-99%] vs. non-XRT: 85.5% +/- 8.7% [range 70%-99%], P = NS). Perioperative outcomes, including the composite 30 day stroke/myocardial infarction/mortality rate did not differ significantly between the two groups (XRT: 0% vs. non-XRT: 3.2%, P = NS). Twelve-month freedom from restenosis rates did not differ significantly at the 50% threshold (XRT: 95.5% vs. non-XRT: 90.3%, P = NS) or at the 70% threshold (XRT: 95.5% vs. non-XRT: 96.5%, P = NS). Target lesion revascularization did not differ significantly (XRT: 0% vs. non-XRT: 0.5%, P = NS). Photomicroscopy demonstrated a trend towards increased particle number and size in the XRT filters, however the results did not achieve statistical significance: particle number (XRT: 9.8 +/- 8.4 vs. non-XRT: 9.6 +/- 11.7, P = NS), %patients with particle size >1000 microm (XRT: 47.4% vs. non-XRT: 30.5%, P = NS). CONCLUSIONS This study suggests that the durability of CAS and the characteristics of captured embolic particles are not altered by a history of neck XRT. This supports the safety and efficacy of CAS for the treatment of patients with a history of neck XRT. Prior neck XRT may predispose the patient to the de novo development of stenoses at locations that were not previously treated.

Direct hydrogel encapsulation of pluripotent stem cells enables ontomimetic differentiation and growth of engineered human heart tissues.

Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue.

Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression.

Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly efficient in vivo delivery of modified mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injection of FLNP containing 1-10 μg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green fluorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expression kinetics after injecting 5 μg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intracoronary administration of 10 μg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injection, and off-target expression was correspondingly higher. Findings were confirmed in a pilot study in pigs using direct myocardial injection as well as percutaneous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanoparticle-enabled approach for highly efficient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.