[Show abstract][Hide abstract] ABSTRACT: Background:
Surgical myectomy and alcohol septal ablation (ASA) aim to decrease left ventricular outflow tract (LVOT) gradient in hypertrophic cardiomyopathy (HCM). Outcome of myectomy beyond 10 years has rarely been described. We describe 20 years of follow-up of surgical myectomy and 5 years of follow-up for ASA performed for obstructive HCM.
We studied 171 patients who underwent myectomy for symptomatic LVOT obstruction between 1972 and 2006. In addition, we studied 52 patients who underwent ASA for the same indication and who declined surgery. Follow-up of New York Heart Association (NYHA) functional class, echocardiographic data, and vital status were obtained from patient records. Mortality rates were compared with expected mortality rates of age- and sex-matched populations.
Surgical myectomy improved NYHA class (2.74±0.65 to 1.54±0.74, p<0.001), reduced resting gradient (67.4±43.4mmHg to 11.2±16.4mmHg, p<0.001), and inducible LVOT gradient (98.1±34.7mmHg to 33.6±34.9mmHg, p<0.001). Similarly, ASA improved functional class (2.99±0.35 to 1.5±0.74, p<0.001), resting gradient (67.1±26.9mmHg to 23.9±29.4mmHg, p<0.001) and provoked gradient (104.4±34.9mmHg to 35.5±38.6mmHg, p<0.001). Survival after myectomy at 5, 10, 15, and 20 years of follow-up was 92.9%, 81.1%, 68.9%, and 47.5%, respectively. Of note, long-term survival after myectomy was lower than for the general population [standardized mortality ratio (SMR)=1.40, p<0.005], but still compared favorably with historical data from non-operated HCM patients. Survival after ASA at 2 and 5 years was 97.8% and 94.7%, respectively. Short-term (5 year) survival after ASA (SMR=0.61, p=0.48) was comparable to that of the general population.
Long-term follow-up of septal reduction strategies in obstructive HCM reveals that surgical myectomy and ASA are effective for symptom relief and LVOT gradient reduction and are associated with favorable survival. While overall prognosis for the community HCM population is similar to the general population, the need for surgical myectomy may identify a sub-group with poorer long-term prognosis. We await long-term outcomes of more extensive myectomy approaches adopted in the past 10 years at major institutions.
No preview · Article · Sep 2014 · Journal of Cardiology
[Show abstract][Hide abstract] ABSTRACT: Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models - both resulting in artery stenosis but each underlying a different pathway of development - are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.
Full-text · Article · Jun 2014 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.
[Show abstract][Hide abstract] ABSTRACT: Rationale:
Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.
To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.
Methods and results:
We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile.
Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism.
[Show abstract][Hide abstract] ABSTRACT: Using meta-analysis of eight independent transplant datasets (236 graft biopsy samples) from four organs, we identified a common rejection module (CRM) consisting of 11 genes that were significantly overexpressed in acute rejection (AR) across all transplanted organs. The CRM genes could diagnose AR with high specificity and sensitivity in three additional independent cohorts (794 samples). In another two independent cohorts (151 renal transplant biopsies), the CRM genes correlated with the extent of graft injury and predicted future injury to a graft using protocol biopsies. Inferred drug mechanisms from the literature suggested that two FDA-approved drugs (atorvastatin and dasatinib), approved for nontransplant indications, could regulate specific CRM genes and reduce the number of graft-infiltrating cells during AR. We treated mice with HLA-mismatched mouse cardiac transplant with atorvastatin and dasatinib and showed reduction of the CRM genes, significant reduction of graft-infiltrating cells, and extended graft survival. We further validated the beneficial effect of atorvastatin on graft survival by retrospective analysis of electronic medical records of a single-center cohort of 2,515 renal transplant patients followed for up to 22 yr. In conclusion, we identified a CRM in transplantation that provides new opportunities for diagnosis, drug repositioning, and rational drug design.
Full-text · Article · Oct 2013 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Aim: In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA (shRNA) interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Methods and Results: Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly Luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electro-coagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or PBS as control. Blood flow recovery (BFR) was monitored using Laser Doppler Perfusion Imaging (LDPI), and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared to PL-Luc. BLI signals in vivo were 4.3x105±3.3x105 (MC-Luc) versus 0.4x105±0.3x105 (PL-Luc) at day 28 (p=0.016). Compared to PL-shPHD2 or PBS, MC-shPHD2 significantly improved BFR, up to 50% from day 3 until day 14 after ischemia-induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin (α-SMA) expression and CD31+ expression, respectively (CD31+: 40.3 MC-shPHD2 versus 18.1 PL-shPHD2 and 11.8 PBS). Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative PCR (qPCR). Finally, Western blot analysis confirmed significant higher levels of HIF-1-alpha protein by MC-shPHD2, compared to PL-shPHD2 and PBS. Conclusions: This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease (PAD). The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved post-ischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.
No preview · Article · Oct 2013 · Human gene therapy
[Show abstract][Hide abstract] ABSTRACT: Background—Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.
Methods and Results—Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell–derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.
Conclusions—We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
[Show abstract][Hide abstract] ABSTRACT: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.
We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ERG521T) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.
Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.
The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.
No preview · Article · Aug 2013 · Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both pre-clinical and clinical evaluations of this therapy are underway, and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bi-directional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging following vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression ( r <sup>2</sup>=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in pre-clinical models of myocardial ischemia.
Full-text · Article · Aug 2013 · Human Gene Therapy Methods
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells (MSCs) have been isolated from many tissues including gestational tissue. To date a study comparing the properties and suitability of these cells in cell-based therapies is lacking. In this study we compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory capabilities of human MSCs derived from umbilical cord lining (CL-MSC), umbilical cord blood (CB-MSC), placenta (P-MSC), and Wharton´s jelly (WJ-MSC). Differences were noted in differentiation, proliferation, and migration, with CL-MSCs showing the highest proliferation and migration rates resulting in prolonged survival in immunodeficient mice. Moreover, CL-MSCs showed a prolongation in survival in xenogeneic BALB/c mice which was attributed to their ability to dampen TH1 and TH2 responses. Weaker human cellular immune responses were detected against CL-MSCs and P-MSCs, which were correlated with their lower HLA I expression. Furthermore HLA II was up-regulated less substantially by CL-MSCs and CB-MSCs after IFN-γ stimulation. MSC types did not differ in indolamine 2,3-dioxygenase (IDO) expression after IFN-γ stimulation. Despite their lower IDO, HLA-G, and TGF-β1 expression, only CL-MSCs were able to reduce the release of IFN-γ by lymphocytes in a mixed lymphocyte reaction. In summary, CL-MSCs showed the best characteristics for cell-based strategies, as they are hypo-immunogenic and show high proliferation and migration rates. In addition, these studies show for the first time that although immunomodulatory molecules HLA-G, HLA-E, and TGF-β play an important role in MSC immune evasion, basal and induced HLA expression seems to be decisive in determining the immunogenicity of MSCs.
No preview · Article · May 2013 · Stem cells and development
[Show abstract][Hide abstract] ABSTRACT: Background:
Lung transplantation and heart-lung transplantation represent surgical options for treatment of medically refractory idiopathic pulmonary arterial hypertension. The effect of the lung allocation score on wait-list and transplantation outcomes in patients with idiopathic pulmonary arterial hypertension is poorly described.
Methods and results:
Adults diagnosed with idiopathic pulmonary arterial hypertension and listed for transplantation in the 80 months before and after the lung allocation score algorithm was implemented (n=1430) were identified in the United Network for Organ Sharing thoracic registry. Patients were stratified by organ listed and pre- and post-lung allocation score era. The cumulative incidences of transplantation and mortality for wait-listed patients in both eras were appraised with competing outcomes analysis. Posttransplantation survival was assessed with the Kaplan-Meier method. These analyses were repeated in propensity-matched subgroups. Cox proportional hazards analysis evaluated the effect of prelisting and pretransplantation characteristics on mortality. We found that patients in the post-lung allocation score era had significantly worse comorbidities; nevertheless, both lung transplantation and heart-lung transplantation candidates in this era enjoyed lower wait-list mortality and a higher incidence of transplantation in unmatched and propensity-matched analyses. On multivariable analysis, heart-lung transplantation and double-lung transplantation were associated with improved survival from the time of wait-listing, as was being listed at a medium- to high-volume institution. Donor/recipient sex matching predicted posttransplantation survival.
The incidence of transplantation has increased while wait-list mortality has decreased in patients with idiopathic pulmonary arterial hypertension wait-listed for transplantation in the post-lung allocation score era. Both heart-lung transplantation and double-lung transplantation are predictive of survival in transplantation candidates with idiopathic pulmonary arterial hypertension, as is being listed at a medium- to high-volume institution. Donor/recipient sex matching is associated with better posttransplantation survival.
[Show abstract][Hide abstract] ABSTRACT: Background:
Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.
Methods and results:
Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.
We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.