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Circulation research [journal]
- Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 21.
Rationale: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. Objective: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. Methods and Results: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared to separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCMs loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiomyocyte markers. Conclusions: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.
- Remnant Cholesterol, Low-Density Lipoprotein Cholesterol, and Blood Pressure as Mediators from Obesity to Ischemic Heart Disease. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 19.
Rationale: Obesity leads to increased ischemic heart disease (IHD) risk, but the risk is thought to be mediated through intermediate variables and may not be caused by increased weight per se. Objective: To test the hypothesis that the increased IHD risk due to obesity is mediated through lipoproteins, blood pressure, glucose, and/or C-reactive protein. Methods and Results: ~90,000 participants from Copenhagen were included in a Mendelian randomization design with mediation analyses. Associations were examined using conventional measurements of body mass index (BMI) and intermediate variables, and using genetic variants associated with these. During up to 22 years of follow-up 13,945 participants developed IHD. The increased IHD risk due to obesity was partly mediated through elevated levels of nonfasting remnant cholesterol and low-density lipoprotein(LDL) cholesterol, through elevated blood pressure, and possibly also through elevated nonfasting glucose levels; however, reduced high-density lipoprotein cholesterol and elevated C-reactive protein levels were not mediators in genetic analyses. The three intermediate variables that explained the highest excess risk of IHD from genetically determined obesity were LDL cholesterol with 8%, systolic blood pressure with 7%, and remnant cholesterol with 7% excess risk of IHD. Corresponding observational excess risks using conventional BMI were 21%, 11%, and 20%, respectively. Conclusions: The increased IHD risk due to obesity was partly mediated through elevated levels of nonfasting remnant and LDL cholesterol, and through elevated blood pressure. Our results suggest there may be benefit to gain by reducing levels of these risk factors in obese individuals not able to achieve sustained weight loss.
- Bone Marrow Characteristics Associated with Changes in Infarct Size after STEMI: A Biorepository Evaluation from the CCTRN TIME Trial. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 18.
Rationale: Despite significant interest in bone marrow mononuclear cell (BMC) therapy for ischemic heart disease, current techniques have resulted in only modest benefits. However, select patients have shown improvements after autologous BMC therapy, but the contributing factors are unclear. Objective: The purpose of this study was to identify BMC characteristics associated with a reduction in infarct size following STEMI. Methods and Results: This prospective study comprised patients consecutively enrolled in the CCTRN TIME trial who agreed to have their BMCs stored and analyzed at the CCTRN Biorepository. Change in infarct size between baseline (3 days after percutaneous coronary intervention) and 6-month follow-up was measured by cardiac magnetic resonance imaging (cMRI). Infarct-size measurements and BMC phenotype and function data were obtained for 101 patients (mean age, 56.5 years; mean screening ejection fraction, 37%; mean baseline cMRI ejection fraction, 45%). At 6 months, 75 patients (74.3%) showed a reduction in infarct size (mean change, -21.0%±17.6%). Multiple regression analysis indicated that infarct size reduction was greater in patients who had a larger percentage of CD31+ BMCs (P=0.046) and in those with faster BMC growth rates in CFU-Hill and ECFC functional assays (P=0.033 and P=0.032, respectively). Conclusions: This study identified BMC characteristics associated with a better clinical outcome in patients with STEMI and highlighted the importance of endothelial precursor activity in regenerating infarcted myocardium. Furthermore, it suggests that for these STEMI patients, myocardial repair was more dependent on baseline BMC characteristics than on whether the patient underwent intracoronary BMC transplantation.
- Intracoronary Autologous Cardiac Progenitor Cell Transfer in Patients with Hypoplastic Left Heart Syndrome (TICAP): A Prospective Phase 1 Controlled Trial. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 17.
Rationale: Hypoplastic left heart syndrome (HLHS) remains a lethal congenital cardiac defect. Recent studies have suggested that intracoronary administration of autologous cardiosphere-derived cells (CDCs) may improve ventricular function. Objective: The aim of this study was to test whether intracoronary delivery of CDCs is feasible and safe in patients with HLHS. Methods and Results: Between January 5, 2011, and January 16, 2012, 14 patients (1.8 ± 1.5 years) were prospectively assigned to receive intracoronary infusion of autologous CDCs 33.4 ± 8.1 days after staged procedures (n=7), followed by 7 controls with standard palliation alone. The primary endpoint was to assess the safety and the secondary endpoint included the preliminary efficacy to verify the right ventricular ejection fraction (RVEF) improvements between baseline and 3 months. Manufacturing and intracoronary delivery of CDCs were feasible and no serious adverse events were reported within the 18-month follow-up. Patients treated with CDCs showed RVEF improvement from baseline to 3-month follow-up (46.9 ± 4.6% to 52.1 ± 2.4%, P=0.008). Compared with controls at 18 months, cMRI analysis of CDC-treated patients showed a higher RVEF (31.5 ± 6.8% vs. 40.4 ± 7.6%, P=0.049), improved somatic growth (P=0.0005), reduced heart failure status (P=0.003), and lower incidence of coil occlusion for collaterals (P=0.007). Conclusions: Intracoronary infusion of autologous CDCs appears to be feasible and safe in children with HLHS after staged surgery. Large phase 2 trials are warranted to examine the potential effects of cardiac function improvements and the long-term benefits of clinical outcomes.
- 2014 AHA Late-Breaking Basic Science Abstracts. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 14.
- CD4+ Natural Killer T Cells Potently Augment Aortic Root Atherosclerosis by Perforin- and Granzyme B-Dependent Cytotoxicity. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 14.
Rationale: CD4(+) Natural Killer T (NKT) cells augment atherosclerosis in ApoE(-/-) mice but their mechanisms of action are unknown. Objective: We investigated the roles of bystander T, B and NK cells, NKT cell derived IFN-γ, IL-4 and IL-21 cytokines and NKT cell derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. Methods and Results: Transfer of CD4(+) NKT cells into T and B cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ~75% that was ~30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T, B and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4+ NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived IFN-γ, IL-4 and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, IFN-γ or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ~95%, ~80% and ~70% respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores and proinflammatory VCAM-1 and MCP-1were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis Conclusions: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme-B dependent apoptosis that increases post-apoptotic necrosis and inflammation.
- Circulating Cells Contribute to Cardiomyocyte Regeneration After Injury. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 14.
Rationale: The contribution of bone marrow-borne hematopoietic cells to the ischemic myocardium has been documented. However, a pivotal study reported no evidence of myocardial regeneration from hematopoietic-derived cells. The study did not take into account the possible effect of early injury-induced signaling as the test mice were parabiotically paired to partners immediately after surgery-induced myocardial injury when cross circulation has not yet developed. Objective: To re-evaluate the role of circulating cells in the injured myocardium. Methods and Results: By combining pulse-chase labeling and parabiosis model, we show that circulating cells derived from the parabiont expressed cardiac-specific markers in the injured myocardium. Genetic fate-mapping also revealed that circulating hematopoietic cells acquired cardiac cell fate by means of cell fusion and transdifferentiation. Conclusions: These results suggest that circulating cells participate in cardiomyocyte regeneration in a mouse model of parabiosis when the circulatory system is fully developed before surgery-induced heart injury.
- GPIHBP1 Missense Mutations Often Cause Multimerization of GPIHBP1 and Thereby Prevent Lipoprotein Lipase Binding. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 11.
Rationale: GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia. Objective: We sought to understand mechanisms by which GPIHBP1 mutations prevent LPL binding and lead to chylomicronemia. Methods and Results: We expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1's Ly6 domain (including mutants identified in chylomicronemia patients) led to the formation of disulfide-linked dimers and multimers. GPIHBP1 dimerization/multimerization was not unique to cysteine mutations; mutations in other amino acid residues, including several associated with chylomicronemia, also led to protein dimerization/multimerization. The loss of GPIHBP1 monomers is quite relevant to the pathogenesis of chylomicronemia because only GPIHBP1 monomers-and not dimers or multimers-are capable of binding LPL. One GPIHBP1 mutant, GPIHBP1-W109S, had distinctive properties. GPIHBP1-W109S lacked the ability to bind LPL but had a reduced propensity for forming dimers or multimers, suggesting that W109 might play a more direct role in binding LPL. In support of that idea, replacing W109 with any of 8 other amino acids abolished LPL binding-and often did so without promoting the formation of dimers and multimers. Conclusions: Many amino acid substitutions in GPIHBP1's Ly6 domain that abolish LPL binding lead to protein dimerization/multimerization. Dimerization/multimerization is relevant to disease pathogenesis, given that only GPIHBP1 monomers are capable of binding LPL.
- Myocardial Ischemia and Reperfusion Leads to Transient CD8 Immune Deficiency and Accelerated Immunosenescence in CMV-Seropositive Patients. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 10.
Rationale: There is mounting evidence of a higher incidence of coronary heart disease (CHD) in cytomegalovirus (CMV) seropositive individuals. Objective: The aim of this study was to investigate whether acute MI triggers an inflammatory T-cell response that might lead to accelerated immunosenescence in CMV-seropositive patients. Methods and Results: Thirty-four patients with acute MI undergoing primary PCI (PPCI) were longitudinally studied within 3 months following reperfusion (Cohort A). In addition, 54 patients with acute and chronic MI were analyzed in a cross-sectional study (Cohort B). CMV-seropositive patients demonstrated a greater fall in the concentration of terminally differentiated CD8 effector memory T cells (TEMRA) in peripheral blood during the first 30 min of reperfusion compared with CMV-seronegative patients (-192 vs. -63 cells/µl; p=0.008), correlating with the expression of programmed cell death-1 (PD-1) before PPCI (r=0.8; p=0.0002). A significant proportion of TEMRA cells remained depleted for at least 3 months in CMV-seropositive patients. Using high-throughput 13-parameter flow cytometry and HLA class I CMV-specific dextramers, we confirmed an acute and persistent depletion of terminally differentiated TEMRA and CMV-specific CD8(+) cells in CMV-seropositive patients. Long-term reconstitution of the TEMRA pool in chronic CMV-seropositive post-MI patients was associated with signs of terminal differentiation including an increase in KLRG1 and shorter telomere length in CD8(+) T cells (2225 bp vs. 3397 bp; p<0.001). Conclusions: Myocardial ischemia and reperfusion in CMV-seropositive patients undergoing PPCI leads to acute loss of antigen-specific, terminally differentiated CD8 T-cells, possibly through PD-1-dependent programmed cell death. Our results suggest that acute MI and reperfusion accelerate immunosenescence in CMV-seropositive patients.
- Iron, Inflammation, and Early Death in Adults with Sickle Cell Disease. [JOURNAL ARTICLE]
- Circ Res 2014 Nov 6.
Rationale: Patients with sickle cell disease (SCD) have markers of chronic inflammation but the mechanism of inflammation and its relevance to patient survival are unknown. Objective: To assess the relationship between iron, inflammation and early death in SCD. Methods and Results: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7 and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4 and interleukin-6 expression were more than 100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared to 14 matched SCD patients with the wild type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months, (IQR 24-82), C-reactive protein was strongly and independently associated with early death (HR 3.0, 95%CI 1.7-5.2, P<0.001). Conclusions: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.