Marc P. Pelletier

Myocardial tissue engineering with autologous myoblast implantation

OBJECTIVEnImplanting myoblasts derived from autologous skeletal muscle, that is, satellite cells, for myocardial replacement has many advantages when compared with implanting either fetal cardiac myocytes (ethical and donor availability issues) or established cell lines (oncogenicity). Furthermore, autologous myoblasts do not require immunosuppression. The feasibility of satellite cell differentiation into muscle fibers, after implantation into the myocardium, was confirmed by means of a unique cell-labeling technique.nnnMETHODSnMyoblasts (satellite cells) isolated from the skeletal muscle of adult rats are labeled with 4,6-diamidino-2-phenylindone, which binds to DNA and to the protein tubulin to form a fluorescent complex, and implanted into the left ventricular wall of isogenic rats. The specimens are harvested 1 to 4 weeks after myoblast implantation. Histologic sections are examined under a fluorescent microscope.nnnRESULTSnThe labeling efficiency of satellite cells with 4,6-diamidino-2-phenylindole is nearly 100%. In 4 specimens, the progressive differentiation of implanted myoblasts into fully developed striated muscle fibers can be observed.nnnCONCLUSIONnOur earlier studies of autologous myoblast implantation into the cryoinjured myocardium of dogs suggested that these cells could differentiate into cardiac myocytes. However, it had been difficult to firmly establish these findings with the use of cell markers, thereby proving that the neomyocardium had indeed been derived from the implanted myoblasts. In this study, using 4,6-diamidino-2-phenylindole as a satellite cell marker, we were able to demonstrate that the implanted satellite cells did in fact differentiate into fully developed, labeled muscle fibers. Because of the obvious advantages of using autologous donor myoblasts, the clinical application of this approach may provide a novel strategy for the future management of heart failure.

Angiogenic Effects Despite Limited Cell Survival of Bone Marrow-Derived Mesenchymal Stem Cells under Ischemia

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.

Thoratec ventricular assist devices in pediatric patients: update on clinical results.

Particularly in pediatric patients, mechanical circulatory support remains a clinical challenge. We analyzed the Stanford experience with use of the Thoratec ventricular assist device (VAD) in children and adolescents and data from the company’s voluntary database. Through January 2005, 209 patients up to 18 years of age have been supported with the Thoratec VAD worldwide. Mean age was 14.5 years (range 5–18 years), mean weight was 57 kg (range 17–118 kg), and mean body surface area was 1.6 m2 (range 0.7–2.3 m2). The majority of patients were supported for cardiomyopathies (55%) and acute myocarditis (25%). A minority (6%) was treated for end-stage congenital heart disease. Average duration of support was 44 days (0–434 days). Overall survival to transplantation or weaning off the device was 68%. Survival rates were higher for patients with cardiomyopathies (74%) and acute myocarditis (86%) compared with patients with congenital heart disease (27%). We performed a subanalysis in small children with a body surface area of less 1.3 m2. This subgroup had a higher incidence of congenital heart disease and a slightly lower survival (52%). Aspects of the particular risks and device management in these small patients are discussed.

Novel Immunosuppression : R348, a JAK3-and Syk-Inhibitor Attenuates Acute Cardiac Allograft Rejection

Background. Janus kinase (JAK)3 is crucial for signal transduction downstream of various cytokine receptors in immune cells. This is the first report on the novel JAK3 inhibitor R348. Methods. (1) Detailed pharmacokinetic data were obtained in rats; (2) multiple in vitro enzyme inhibition assays were performed to characterize the drug; (3) prevention of acute rejection was investigated in animals treated with different doses of R348 or rapamycin for 5 days; and (4) cardiac allograft survival after a 10-day treatment period was studied for various regimens of R348, tacrolimus, or rapamycin; combination indices were calculated to evaluate drug interactions. Results. (1) Plasma levels of R348s active metabolite R333 sustained high for 8 hr or more, depending on the dose. (2) In vitro enzyme assays showed potent inhibition of JAK3- and Syk-dependent pathways. (3) R348 40 mg/kg preserved graft function, significantly reduced graft infiltration, and decreased histologic ISHLT rejection scores on postoperative day 5. Results were similar to those of rapamycin 3 mg/kg. Likewise, both drugs significantly reduced the cellular Th1 and Th2 immune responses, as determined by enzyme-linked immunosorbent assays. Intragraft inflammatory cytokine upregulation was similarly suppressed by R348 and rapamycin. R348 10 mg/kg was subtherapeutic. (4) Allograft survival was similar for R348 20 and 40 mg/kg, which was comparable with therapeutically dosed tacrolimus or rapamycin. In combination regimens, R348 demonstrated highly beneficial synergistic interactions with tacrolimus. Conclusions. R348 is a promising novel JAK3/Syk-inhibitor with favorable pharmacokinetics and biological activity. It effectively diminishes acute cardiac allograft rejection and is suitable for combination regimens with tacrolimus.

StructuralAspirin Versus Aspirin Plus Clopidogrel as Antithrombotic Treatment Following Transcatheter Aortic Valve Replacement With a Balloon-Expandable Valve: The ARTE (Aspirin Versus Aspirin + Clopidogrel Following Transcatheter Aortic Valve Implantation) Randomized Clinical Trial

OBJECTIVESnThe aim of this study was to compare aspirin plus clopidogrel with aspirin alone as antithrombotic treatment following transcatheter aortic valve replacement (TAVR) for the prevention of ischemic events, bleeding events, and death.nnnBACKGROUNDnFew data exist on the optimal antithrombotic therapy following TAVR.nnnMETHODSnThis was a randomized controlled trial comparing aspirin (80 to 100 mg/day) plus clopidogrel (75 mg/day) (dual antiplatelet therapy [DAPT]) versus aspirin alone (single-antiplatelet therapy [SAPT]) in patients undergoing TAVRxa0with a balloon-expandable valve. The primary endpoint was the occurrence of death, myocardial infarction (MI), stroke or transient ischemic attack, or major or life-threatening bleeding (according to Valve Academic Research Consortium 2 definitions) within the 3 months following the procedure. The trial was prematurely stopped after the inclusion of 74% of the planned study population.nnnRESULTSnA total of 222 patients were included, 111 allocated to DAPT and 111 to SAPT. The composite of death, MI, stroke or transient ischemic attack, or major or life-threatening bleeding tended to occur more frequently in the DAPT group (15.3% vs.xa07.2%, pxa0= 0.065). There were no differences between groups in the occurrence of death (DAPT, 6.3%; SAPT, 3.6%; pxa0= 0.37), MI (DAPT, 3.6%; SAT, 0.9%; pxa0= 0.18), or stroke or transient ischemic attack (DAPT, 2.7%; SAPT, 0.9%; pxa0= 0.31) at 3 months. DAPT was associated with a higher rate of major or life-threatening bleeding events (10.8% vs. 3.6% in the SAPT group, pxa0=xa00.038). There were no differences between groups in valve hemodynamic status post-TAVR.nnnCONCLUSIONSnThis small trial showed that SAPT (vs. DAPT) tended to reduce the occurrence of major adverse events following TAVR. SAPT reduced the risk for major or life-threatening events while not increasing the risk for MI or stroke. Larger studies are needed to confirm these results. (Aspirin Versus Aspirinxa0+ Clopidogrel Following Transcatheter Aortic Valve Implantation: The ARTE Trial [ARTE], NCT01559298; Aspirin Versus Aspirin+Clopidogrel as Antithrombotic Treatment Following TAVI [ARTE], NCT02640794).

Multimodal evaluation of in vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells.

OBJECTIVEnMouse embryonic stem cells have demonstrated potential to restore infarcted myocardium after acute myocardial infarction. Although the underlying mechanism remains controversial, magnetic resonance imaging has provided reliable in vivo assessment of functional recovery after cellular transplants. Multimodal comparison of the restorative effects of mouse embryonic stem cells and mouse embryonic fibroblasts was performed to validate magnetic resonance imaging data and provide mechanistic insight.nnnMETHODSnSCID-beige mice (n = 55) underwent coronary artery ligation followed by injection of 2.5 x 10(5) mouse embryonic stem cells, 2.5 x 10(5) mouse embryonic fibroblasts, or normal saline solution. In vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells was evaluated by (1) in vivo pressure-volume loops, (2) in vivo bioluminescence imaging, and (3) ex vivo TaqMan (Roche Molecular Diagnostics, Pleasanton, Calif) polymerase chain reaction and immunohistologic examination.nnnRESULTSnIn vivo magnetic resonance imaging demonstrated significant improvement in left ventricular ejection fraction at 1 week in the mouse embryonic stem cell group. This finding was validated with (1) pressure-volume loop analysis demonstrating significantly improved systolic and diastolic functions, (2) bioluminescence imaging and polymerase chain reaction showing superior posttransplant survival of mouse embryonic stem cells, (3) immunohistologic identification of cardiac phenotype within engrafted mouse embryonic stem cells, and (4) polymerase chain reaction measuring increased expressions of angiogenic and antiapoptotic genes and decreased expressions of antifibrotic genes.nnnCONCLUSIONnThis study validates in vivo magnetic resonance imaging as an effective means of evaluating the restorative potential of mouse embryonic stem cells.

The impact of calcium volume and distribution in aortic root injury related to balloon-expandable transcatheter aortic valve replacement

BACKGROUNDnA detailed assessment of calcium within the aortic root may provide important additional information regarding the risk of aortic root injury during transcatheter heart valve replacement (TAVR).nnnOBJECTIVEnWe sought to delineate the effect of calcium volume and distribution on aortic root injury during TAVR.nnnMETHODSnThirty-three patients experiencing aortic root injury during TAVR with a balloon-expandable valve were compared with a control group of 153 consecutive TAVR patients without aortic root injury (as assessed by post-TAVR multidetector CT). Using commercial software to analyze contrast-enhanced pre-TAVR CT scans, calcium volume was determined in 3 regions: (1) the overall left ventricular outflow tract (LVOT), extending 10xa0mm down from the aortic annulus plane; (2) the upper LVOT, extending 2xa0mm down from the annulus plane; and (3) the aortic valve region.nnnRESULTSnCalcium volumes in the upper LVOT (median, 29 vs 0xa0mm(3); P < .0001) and overall LVOT (median, 74 vs 3xa0mm(3); Pxa0= .0001) were higher in patients who experienced aortic root injury compared with the control group. Calcium in the aortic valve region did not differ between groups. Upper LVOT calcium volume was more predictive of aortic root injury than overall LVOT calcium volume (area under receiver operating curve [AUC], 0.78; 95% confidence interval, 0.69-0.86 vs AUC, 0.71; 95% confidence interval, 0.62-0.82; Pxa0= .010). Upper LVOT calcium below the noncoronary cusp was significantly more predictive of aortic root injury compared to calcium underneath the right coronary cusp or the left coronary cusp (AUC, 0.81 vs 0.68 vs 0.64). Prosthesis oversizing >20% (likelihood ratio test, Pxa0= .028) and redilatation (likelihood ratio test, Pxa0= .015) improved prediction of aortic root injury by upper LVOT calcium volume.nnnCONCLUSIONnCalcification of the LVOT, especially in the upper LVOT, located below the noncoronary cusp and extending from the annular region, is predictive of aortic root injury during TAVR with a balloon-expandable valve.

Introducing the first polymer-free leflunomide eluting stent.

BACKGROUNDnWe here describe the pharmacological characteristic, in vivo efficacy, and in vitro mechanisms of a polymer-free leflunomide eluting stent in comparison to its rapamycin-coated equivalent.nnnMETHODSnStents were coated with 40 mM solutions of leflunomide (L) or rapamycin (R) or were left uncoated (BM). Neointima formation was assessed 6 weeks after implantation into Sprague Dawley rats by optical coherence tomographies (OCT) and histopathology. In vitro proliferation assays were performed using isolated endothelial and smooth-muscle-cells from Sprague Dawley rats to investigate the cell-specific pharmacokinetic effect of leflunomide and rapamycin.nnnRESULTSnHPLC-based drug release kinetics revealed a similar profile with 90% of the drug being released after 12.1+/-0.2 (L) and 13.0+/-0.2 days (R). After 6 weeks, OCTs showed that in-stent luminal obliteration was less for the coated stents (L:12.0+/-9.4%, R:13.3+/-13.1%) when compared to identical bare metal stents (BM:26.4+/-4.7%; p<or=0.046). Histology with computer-assisted morphometry was performed and demonstrated reduced in-stent I/M thickness ratios (L:2.5+/-1.2, R:3.7+/-3.3, BM:6.7+/-2.3, p<or=0.049 for L and R vs. BM) and neointimal areas (L:0.6+/-0.3, R:0.7+/-0.2, BM:1.3+/-0.4, p<or=0.039 for L and R vs. BM) with stent coating. No differences were found for injury and inflammation scores (L and R vs. BM; p=NS). In vitro SMC proliferation was dose-dependently and similarly inhibited by L and R at 1-100 nM (p=NS L vs. R). Interestingly, human EC proliferation at 10-100 nM was significantly inhibited only by R (p<0.001), but not by L (p=NS).nnnCONCLUSIONSnThe diminished inhibition of EC proliferation may improve arterial healing and contribute to the safety profile of the leflunomide stent.

Effect of inhaled tacrolimus on cellular and humoral rejection to prevent posttransplant obliterative airway disease

This study aimed to investigate the pharmacokinetics after tacrolimus aerosol inhalation and to assess its efficacy to suppress acute and chronic airway allograft rejection. Orthotopic tracheal transplantations were performed and tacrolimus (4 mg/kg) was administered orally (PO) or via aerosol (AER). Tracheal tissue level AUCs0–12 were similar in both treatment groups, but blood AUCs0–12 were approximately 5.5‐fold lower with AER (p < 0.001). Interestingly, only PO animals showed elevated BUN, cholesterol and triglycerides on POD 60 (p < 0.05). Histology of grafts harvested after 6 and 60 days revealed that both treatment groups were similarly effective in suppressing graft mononuclear infiltration (p < 0.001). Cellular immune activation (assessed by IFN‐γ‐ and IL‐4‐ELISPOTS), however, was far more effectively suppressed by tacrolimus PO (p < 0.001). In both treatment groups, the vigorous alloreactive IgM‐antibody surge was effectively inhibited (p < 0.001). Due to the insufficient systemic cellular immunosuppression, discontinuation of tacrolimus AER resulted in a far stronger (3.5‐fold) graft infiltration on POD 8 compared to PO (p < 0.001). Tacrolimus aerosol reduces systemic side effects and effectively protects the airway graft from early cellular rejection and chronic obliterative airway disease.

Coronary atherosclerosis in cardiac transplant patients treated with total lymphoid irradiation

BACKGROUNDnMultiple episodes of rejection following cardiac transplantation have been associated with an increased incidence of coronary atherosclerosis. Total lymphoid irradiation (TLI) has been shown to be a successful treatment for persistent allograft rejection, but its effect on coronary arterial disease has yet to be evaluated.nnnMETHODSnFrom 1987 to 1999, 40 patients required TLI for persistent or recurrent allograft rejection following heart transplantation. Each patients (Group 1, n = 31) post-transplant coronary angiograms were examined and compared with those of a control group (Group 2, (n = 32) matched for time of transplantation. Degree of coronary stenosis was assessed on a 6-point scale. All patients received induction therapy (rabbit anti-thymocyte globulin or OKT3) and standard triple immunosuppressive therapy. TLI (80 cGy x 10 fractions) was used for the treatment of recurrent or persistent rejection on the basis of clinical indications. Actuarial survival, number and treatment of rejection episodes, and severity of coronary artery disease were compared in each group.nnnRESULTSnRecipient gender, age, race and cytomegalovirus (CMV) status at time of transplant, along with donor gender, CMV status and graft ischemia time, were similar in both groups. Group 1 donor age was younger than that of Group 2 (22.2 +/- 11.2 vs 31.5 +/- 13.6 years, p = 0.004), and the indication for surgery in Group 1 patients was more likely to be ischemic heart disease (15 of 31 vs 6 of 32, p = 0.02). Mean follow-up was 5.7 +/- 3.5 years in Group 1 vs 6.9 +/- 3.8 in Group 2 (p = NS). Group 1 had more rejection episodes (4.4 +/- 2.2 vs 2.3 +/- 2.0, p = 0.0002) and more steroid treatments (9.78 +/- 4.0 g vs 5.14 +/- 4.7 g, p < 0.0001), but less coronary artery disease compared with Group 2 (p = 0.035).nnnCONCLUSIONSnDespite multiple episodes of rejection, patients treated with TLI after cardiac transplant appear to develop less coronary atherosclerosis than appropriately matched controls.