Valve calcification and inflammation play key roles in the development of aortic stenosis (AS). The Wnt pathways have been linked to inflammation, bone metabolism, angiogenesis, and heart valve formation. We hypothesized that soluble Wnt modulators may be dysregulated in symptomatic AS.
Methods and Results
We measured circulating levels (n=136) and aortic valve tissue expression (n=16) of the secreted Wnt modulators secreted frizzled related protein-3, dickkopf-1 (DKK-1), and Wnt inhibitory factor-1 (WIF-1) by enzyme immunoassay, immunostaining, and RT-PCR in patients with symptomatic, severe AS and investigated associations with echocardiographic parameters of AS and cardiac function. Finally, we assessed the prognostic value of these Wnt modulators in relation to all-cause mortality (n=35) during long-term follow-up (median 4.6 years; survivors, 4.8 years; nonsurvivors, 1.9 years) in these patients. Our main findings were: (1) serum levels of all Wnt modulators were markedly elevated in patients with symptomatic AS (mean increase 231% to 278%, P<0.001), (2) all Wnt modulators were present in calcified aortic valves but correlated poorly with systemic levels or degree of AS, (3) some modulators (ie, WIF-1) were associated with the degree of myocardial function and valvular calcification, (4) all Wnt modulators, and DKK-1 in particular, predicted long-term mortality in these patients also after adjusting for conventional predictors including NT-proBNP.
Together, these in vivo data support the involvement of Wnt signaling in the development of AS and suggest that circulating Wnt modulators should be further investigated as risk markers in larger AS populations, including patients with asymptomatic disease.
aortic stenosis; mortality; prognosis; Wnt
Data are sparse regarding the long-term association of favorable levels of all major cardiovascular disease risk factors (RFs) (ie, low risk [LR]) with ankle-brachial index (ABI).
Methods and Results
In 2007–2010, the Chicago Healthy Aging Study reexamined a subset of participants aged 65 to 84 years from the Chicago Heart Association Detection Project in Industry cohort (baseline examination, 1967–1973). RF groups were defined as LR (untreated blood pressure ≤120/≤80 mm Hg, untreated serum cholesterol <200 mg/dL, body mass index <25 kg/m2, not smoking, no diabetes) or as 0 RFs, 1 RF, or 2+ RFs based on the presence of blood pressure ≥140/≥90 mm Hg or receiving treatment, serum cholesterol ≥240 mg/dL or receiving treatment, body mass index ≥30 kg/m2, smoking, or diabetes. ABI at follow-up was categorized as indicating PAD present (≤0.90), as borderline PAD (0.91 to 0.99), or as normal (1.00 to 1.40). We included 1346 participants with ABI ≤1.40. After multivariable adjustment, the presence of fewer baseline RFs was associated with a lower likelihood of PAD at 39-year follow-up (P for trend is <0.001). Odds ratios (95% CIs) for PAD in persons with LR, 0 RFs, or 1 RF compared with those with 2+ RFs were 0.14 (0.05 to 0.44), 0.28 (0.13 to 0.59), and 0.33 (0.16 to 0.65), respectively; findings were similar for borderline PAD (P for trend is 0.005). The association was mainly due to baseline smoking status, cholesterol, and diabetes. Remaining free of adverse RFs or improving RF status over time was also associated with PAD.
LR profile in younger adulthood (ages 25 to 45) is associated with the lowest prevalence of PAD and borderline PAD 39 years later.
aging; atherosclerosis; cardiovascular disease; peripheral artery disease; risk factors
Vascular calcification resembles bone formation and involves vascular smooth muscle cell (SMC) transition to an osteoblast‐like phenotype to express Runx2, a master osteoblast transcription factor. One possible mechanism by which Runx2 protein expression is induced is downregulation of inhibitory microRNAs (miR).
Methods and Results
Human coronary artery SMCs (CASMCs) treated with bone morphogenetic protein‐2 (BMP‐2; 100 ng/mL) demonstrated a 1.7‐fold (P<0.02) increase in Runx2 protein expression at 24 hours. A miR microarray and target prediction database analysis independently identified miR‐30b and miR‐30c (miR‐30b‐c) as miRs that regulate Runx2 expression. Real‐time–polymerase chain reaction confirmed that BMP‐2 decreased miR‐30b and miR‐30c expression. A luciferase reporter assay verified that both miR‐30b and miR‐30c bind to the 3′‐untranslated region of Runx2 mRNA to regulate its expression. CASMCs transfected with antagomirs to downregulate miR‐30b‐c demonstrated significantly increased Runx2, intracellular calcium deposition, and mineralization. Conversely, forced expression of miR‐30b‐c by transfection with pre–miR‐30b‐c prevented the increase in Runx2 expression and mineralization of SMCs. Calcified human coronary arteries demonstrated higher levels of BMP‐2 and lower levels of miR‐30b than did noncalcified donor coronary arteries.
BMP‐2 downregulates miR‐30b and miR‐30c to increase Runx2 expression in CASMCs and promote mineralization. Strategies that modulate expression of miR‐30b and miR‐30c may influence vascular calcification.
microRNA; Runx2; smooth muscle cells; vascular calcification
Evidence-based randomized clinical trials have shown significant benefit of statin treatment with regard to cardiovascular disease. In anticipation of the National Cholesterol Education Program Adult Treatment Panel IV guidelines, we wanted to assess the current state of lipid goal attainment in the high-risk secondary prevention population in the United States. The objectives of the study were to estimate the proportion of high-risk patients treated with statin monotherapy who achieved Adult Treatment Panel III–recommended low-density lipoprotein cholesterol (LDL-C) goals (<100 mg/dL; optional <70 mg/dL) as well as non–high-density lipoprotein cholesterol goals (<130 mg/dL; optional <100 mg/dL).
Methods and Results
This is a cross-sectional, retrospective study of 3 data sources: electronic medical records (2003–September 2010), administrative claims data (2003–2010), and National Health and Nutrition Examination Survey data (2007–2008). High-risk patients (≥18 years of age) were defined as those with a history of coronary heart disease or coronary heart disease risk equivalent who had the latest complete lipid panel measurement and had been treated with statin monotherapy for >90 days at the time of the lipid panel. Cardiovascular disease, coronary heart disease, and coronary heart disease risk equivalents were defined on the basis of availability, specific to each data source. Across the 3 data sources, 20% to 26% of high-risk patients treated with statin monotherapy for >90 days had LDL-C <70 mg/dL, and 67% to 77% had LDL-C <100 mg/dL. The percentages of those attaining both LDL-C goals and non–high-density lipoprotein cholesterol goals were quantitatively smaller (13.5% to 19.0% and 46% to 70%).
Across the 3 data sources, there was consistency in the proportion of high-risk patients treated with statin monotherapy who were at LDL-C goal. A significant number of these statin-treated patients had additional dyslipidemias.
coronary heart disease; dyslipidemia; low-density lipoprotein cholesterol; non–high-density lipoprotein cholesterol; statins
Although controversial, paradoxical embolism via patent foramen ovale (PFO) may account for some of the migraine attacks in a subset of migraine with aura (MA) patients. Induction of MA attacks with air bubble injection during transcranial Doppler ultrasound in MA patients with PFO supports this view. It is likely that cerebral embolism in patients with right-to-left shunt induces bioelectrical abnormalities to initiate MA under some conditions.
Methods and Results
We investigated changes in cerebral bioelectrical activity after intravenous microbubble injection in 10 MA patients with large PFO and right-to-left cardiac shunt. Eight PFO patients without migraine but with large right-to-left shunt and 12 MA patients without PFO served as controls. Four MA patients with PFO were reexamined with sham injections of saline without microbubbles. Bioelectrical activity was evaluated using spectral electroencephalography and, passage of microbubbles through cerebral arteries was monitored with transcranial Doppler ultrasound. Microbubble embolism caused significant electroencephalographic power increase in MA+PFO patients but not in control groups including the sham-injected MA+PFO patients. Headache developed in 2 MA with PFO patients after microbubble injection.
These findings demonstrate that air microembolism through large PFOs may cause cerebral bioelectrical disturbances and, occasionally, headache in MA patients, which may reflect an increased reactivity of their brain to transient subclinical hypoxia–ischemia, and suggest that paradoxical embolism is not a common cause of migraine but may induce headache in the presence of a large PFO and facilitating conditions.
cerebral ischemia; embolism; migraine; PFO; shunts
fractional flow reserve; intravascular ultrasound; left main stenosis; stent
Matrix metalloproteinase-12 (MMP-12) promotes atherosclerosis in animal models. MMP-12 is expressed in only a subset of foam-cell macrophages (FCMs) in human plaques. We investigated whether the prevalence of this MMP-12–expressing subpopulation is a prognostic indicator of adverse outcome in patients after carotid endarterectomy (CEA).
Methods and Results
Serial sections of culprit lesions from 236 patients who underwent CEA and had undergone 3 years of clinical follow-up were stained immunocytochemically for MMP-12 and for CD68, and the MMP-12/CD68 ratio was used to quantify the MMP-12–expressing subpopulation. A high MMP-12/CD68 ratio correlated with a high content of lipid and total macrophages and a low content of vascular smooth muscle cells, as well as with MMP-8 (R=0.211, P=0.001), MMP-9 (R=0.251, P<0.001), and cleaved caspase-3 (R=0.142, P=0.036) activity measured in a neighboring segment. Dual immunohistochemical examination confirmed the location of MMP-12 in a subpopulation of MMP-8– and MMP-9–positive FCMs, whereas all apoptotic FCMs were MMP-12 positive. Patients who yielded plaques within the highest quartile compared with the lowest quartile of MMP-12/CD68 ratio had a 2.4-fold (hazard ratio, 2.4; 95% CI, 1.1- to 5.1-fold; adjusted P=0.027) increased risk of major adverse cardiovascular event and a 3.4-fold (3.4; 1.2- to 9.6-fold, P=0.024) increased risk for stroke.
The prevalence of an MMP-12–positive subset of FCMs is a prognostic marker for adverse clinical outcome after CEA.
atherosclerosis; macrophages; MMP-12; outcome
Growth differentiation factor (GDF)‐15 is a distant and divergent member of the transforming growth factor‐β superfamily (TGF‐β) . There is growing evidence indicating the involvement of GDF‐15 in various pathologies. Expression of GDF‐15 is induced under conditions of inflammation and increased GDF‐15 serum levels are suggested as a risk factor for cardiovascular diseases.
Methods and Results
We show here that GDF‐15 and proinflammatory cytokine interleukin (IL)‐6 levels are highly increased (5‐fold) in cultured oxidized low‐density lipoproteins–stimulated peritoneal macrophages derived from GDF‐15+/+/apolipoprotein (apo) E−/−, mice. Notably, IL‐6 induction on oxidized low‐density lipoproteins stimulation is completely abolished in the absence of GDF‐15. Consistent with our in vitro data GDF‐15 mRNA expression and protein levels are upregulated (2.5‐ to 6‐fold) in the atherosclerotic vessel wall of GDF‐15+/+/apoE−/− mice after a cholesterol‐enriched diet. GDF‐15 deficiency inhibits lumen stenosis (52%) and 18FDG uptake (34%) in the aortic arch despite increased serum triglyceride/cholesterol levels and elevated body weight. Immunohistomorphometric investigations of atherosclerotic lesions reveal a decreased percentage of inflammatory CD11b+ (57%) or IL‐6+, leukocytes, and apoptotic cells (74%) after 20 weeks. However, the total number of macrophages and cell density in atherosclerotic lesions of the innominate artery are increased in GDF‐15−/−/apoE−/− mice.
Our data suggest that GDF‐15 is involved in orchestrating atherosclerotic lesion progression by regulating apoptotic cell death and IL‐6–dependent inflammatory responses to vascular injury.
atherosclerosis; GDF‐15; inflammation; interleukins
Macrophages are critical contributors to atherosclerosis. Single-walled carbon nanotubes (SWNTs) show promising properties for cellular imaging and thermal therapy, which may have application to vascular macrophages.
Methods and Results
In vitro uptake and photothermal destruction of mouse macrophage cells (RAW264.7) were performed with SWNTs (14.7 nmol/L) exposed to an 808-nm light source. SWNTs were taken up by 94±6% of macrophages, and light exposure induced 93±3% cell death. In vivo vascular macrophage uptake and ablation were then investigated in carotid-ligated FVB mice (n=33) after induction of hyperlipidemia and diabetes. Two weeks postligation, near-infrared fluorescence (NIRF) carotid imaging (n=12) was performed with SWNT-Cy5.5 (8 nmol of Cy5.5) given via the tail vein. Photothermal heating and macrophage apoptosis were evaluated on freshly excised carotid arteries (n=21). NIRF of SWNTs showed higher signal intensity in ligated carotids compared with sham, confirmed by both in situ and ex vivo NIRF imaging (P<0.05, ligation versus sham). Immunofluorescence staining showed colocalization of SWNT-Cy5.5 and macrophages in atherosclerotic lesions. Light (808 nm) exposure of freshly excised carotids showed heating and induction of macrophage apoptosis in ligated left carotid arteries with SWNTs, but not in control groups without SWNTs or without light exposure.
Carbon nanotubes accumulate in atherosclerotic macrophages in vivo and provide a multifunctional platform for imaging and photothermal therapy of vascular inflammation.
atherosclerosis; imaging; inflammation; macrophage; photothermal therapy
Thoracic periaortic adipose tissue (TAT) is associated with atherosclerosis and cardiovascular disease (CVD) risk factors and may play a role in obesity‐mediated vascular disease. We sought to determine the prevalence, distribution, and risk factor correlates of high TAT.
Methods and Results
Participants from the Framingham Heart Study (n=3246, 48% women, mean age 51.1 years) underwent multidetector computed tomography; high TAT and visceral adipose tissue (VAT) were defined on the basis of sex‐specific 90th percentiles in a healthy referent sample. The prevalence of high TAT was 38.1% in women and 35.7% in men. Among individuals without high VAT, 10.1% had high TAT. After adjustment for age and VAT, both women and men with high TAT in the absence of high VAT were older and had a higher prevalence of CVD (P<0.0001) compared with those without high TAT. In addition, men in this group were more likely to be smokers (P=0.02), whereas women were more likely to have low high‐density lipoprotein cholesterol (P=0.005).
Individuals in our community‐based sample with high TAT in the absence of high VAT were characterized by an adverse cardiometabolic profile. This adipose tissue phenotype may identify a subset of individuals with distinct metabolic characteristics.
body fat distribution; obesity; perivascular adipose tissue; risk factors; visceral adipose tissue
Central artery dilation and remodeling are associated with higher heart failure and cardiovascular risks. However, data regarding carotid artery diameter from hypertension to heart failure have remained elusive. We sought to investigate this issue by examining the association between carotid artery diameter and surrogates of ventricular dysfunction.
Methods and Results
Two hundred thirteen consecutive patients including 49 with heart failure and preserved ejection fraction (HFpEF), 116 with hypertension, and an additional 48 healthy participants underwent comprehensive echocardiography and tissue Doppler imaging. Ultrasonography of the common carotid arteries was performed for measurement of intima‐media thickness and diameter (CCAD). Cardiac mechanics, including LV twist, were assessed by novel speckle‐tracking software. A substantial graded enlargement of CCAD was observed across all 3 groups (6.8±0.6, 7.7±0.73, and 8.7±0.95 mm for normal, hypertension, and HFpEF groups, respectively; ANOVA P<0.001) and correlated with serum brain natriuretic peptide level (R2=0.31, P<0.001). Multivariable models showed that CCAD was associated with increased LV mass, LV mass‐to‐volume ratio (β‐coefficient=10.9 and 0.11, both P<0.001), reduced LV longitudinal and radial strain (β‐coeffficient=0.81 and −3.1, both P<0.05), and twist (β‐coefficient=−0.84, P<0.05). CCAD set at 8.07 mm as a cut‐off had a 77.6% sensitivity, 82.3% specificity, and area under the receiver operating characteristic curves (AUROC) of 0.86 (95% CI 0.80 to 0.92) in discriminating HFpEF. In addition, CCAD superimposed on myocardial deformation significantly expanded AUROC (for longitudinal strain, from 0.84 to 0.90, P of ΔAUROC=0.02) in heart failure discrimination models.
Increased carotid artery diameter is associated with worse LV geometry, higher brain natriuretic peptide level, and reduced contractile mechanics in individuals with HFpEF.
cardiac mechanics; carotid artery diameter; heart failure; hypertension; remodeling; strain
Hyperglycemia is an independent risk factor for the development of vascular diabetic complications, which are characterized by endothelial dysfunction and tissue‐specific aberrant angiogenesis. Tumor growth is also dependent on angiogenesis. Diabetes affects several cancers in a tissue‐specific way. For example, it positively correlates with the incidence of breast cancer but negatively correlates with the incidence of prostate cancer. The tissue‐specific molecular mechanisms activated by hyperglycemia that control angiogenesis are unknown. Here we describe a novel tissue‐ and cell‐specific molecular pathway that is activated by high glucose and regulates angiogenesis.
Methods and Results
We have identified microRNA 467 (miR‐467) as a translational suppressor of thrombospondin‐1 (TSP‐1), a potent antiangiogenic protein that is implicated in the pathogenesis of several diabetic complications. miR‐467 was upregulated by hyperglycemia in a tissue‐specific manner. It was induced by high glucose in microvascular endothelial cells and in breast cancer cells, where it suppressed the production of TSP‐1 by sequestering mRNA in the nonpolysomal fraction. Mutation of the miR‐467 binding site in TSP‐1 3′ UTR or miR‐467 inhibitor relieved the translational silencing and restored TSP‐1 production. In in vivo angiogenesis models, miR‐467 promoted the growth of blood vessels, and TSP‐1 was the main mediator of this effect. Breast cancer tumors showed increased growth in hyperglycemic mice and expressed higher levels of miR‐467. The antagonist of miR‐467 prevented the hyperglycemia‐induced tumor growth.
Our results demonstrate that miR‐467 is implicated in the control of angiogenesis in response to high glucose, which makes it an attractive tissue‐specific potential target for therapeutic regulation of aberrant angiogenesis and cancer growth in diabetes.
complications of diabetes; microRNA; thrombospondin‐1; translational regulation
Periaortic fat, because of its contiguity with the aorta, may promote vascular remodeling and aortic dilatation. However, the relations between perioartic fat depots and aortic dimensions have not been previously described.
Methods and Results
A total of 3001 individuals (mean age 50±10 years, 49% women) from the Framingham Offspring and Third Generation cohorts underwent computed tomography for quantification of periaortic fat and aortic dimensions. We estimated the association between quantitative periaortic and visceral adipose tissue volumes (per standard deviation [SD] increment of volume) with aortic dimensions in both the thorax and abdomen. Thoracic periaortic fat was associated with higher thoracic aortic dimensions (β coefficient per SD of fat volume 0.67 mm, 95% confidence interval 0.58 to 0.76 mm; P<0.001). The association persisted after adjustment for age, sex, and cardiovascular risk factors including body mass index and visceral adipose tissue volume. Results for the association of periaortic fat and abdominal aortic dimensions were similar. Further adjustment for adipokines (resistin and adiponectin) had no significant impact on these associations.
Periaortic fat volume was associated with aortic dimensions in both the thorax and abdomen, supporting the notion that local fat depots may contribute to aortic remodeling. Further work to understand the mechanisms underlying this association is warranted.
adipose tissue; aneurysm; aorta; peripheral vascular disease
With ideal cardiovascular health metrics, the American Heart Association established a goal of improving cardiovascular health for all Americans by 20% by 2020. Determining how the metrics vary by state is important to the individual states as well as to researchers and policy makers nationwide.
Methods and Results
Using 2009 data from Behavioral Risk Factor Surveillance System, a state‐based telephone survey with 356 441 eligible participants, we examined the 7 metrics defined by the American Heart Association: hypertension, high cholesterol, smoking, body mass index, diabetes, physical activity, and consumption of fruits and vegetables. The 3 primary outcomes of this study were (1) the percentage of the population achieving ideal health status on all 7 factors, (2) the percentage of the population with only 0 to 2 of the 7 metrics (poor cardiovascular health); and (3) the mean overall score (number of ideal metrics). Overall, 3.3% of population was in ideal cardiovascular health, and 9.9% was in poor cardiovascular health. The mean overall score was 4.42. The percentage with ideal cardiovascular health varied from 1.2% (Oklahoma) to 6.9% (District of Columbia ). The adjusted prevalence ratio of ideal cardiovascular health ranged from 0.38, 95% confidence interval 0.29 to 0.52 (Oklahoma), to 1.91, 95% confidence interval 1.51 to 2.42 (District of Columbia), with Illinois as the referent.
In the United States, the cardiovascular health status of the population varies substantially by state. The estimates here could help state programs charged with preventing heart disease and stroke to set their goals for reducing risk and improving cardiovascular health in their jurisdictions.
cardiovascular health; epidemiology; states
editorials; angiogenesis; glucose; tumor
Plasma fibulin-1 levels have been associated with N-terminal pro–B-type natriuretic peptide levels and left atrial size and shown to be predictive of mortality in patients with diabetes. The mechanisms behind these connections are not fully understood but are probably related to its roles as an extracellular matrix protein in cardiovascular tissues.
Methods and Results
One hundred twenty-five patients with severe aortic stenosis who were scheduled for aortic valve replacement (AVR) were evaluated with preoperative echocardiography and their plasma fibulin-1 levels were determined with ELISA. The cohort was followed for a median of 4 years after AVR. Increased restrictive left ventricular (LV) filling pattern was observed with increased plasma fibulin-1 levels (2% versus 29% versus 24% in low, middle, and high plasma fibulin-1 tertile groups, P=0.004). Likewise, reduced longitudinal systolic LV function (6.6±1.1 versus 6.1±1.3 versus 5.7±1.5 cm/s, P=0.05) and increased LV filling pressures was systolic velocity of the mitral annulus observed with increasing plasma fibulin-1 concentrations (ratio of early transmitral flow velocity to early diastolic flow velocity of the mitral annulus 13±4 versus 15±5 versus 16±6 in the fibulin-1 tertile groups, P=0.04).
In patients with symptomatic severe aortic stenosis undergoing AVR, plasma fibulin-1 is associated with restrictive filling of the LV, decreased longitudinal systolic function of the LV, and increased LV filling pressures.
Clinical Trial Registration
URL: http://www.clinicaltrial.gov with Identifier: NCT00294775
aortic valve stenosis; biomarkers; diastolic function; echocardiography
Cholesterol efflux from cells to apolipoprotein A-I (apoA-I) acceptors via the ATP-binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. MicroRNA (miR)-33 is known to target ABCA1 and ABCG1 in vivo.
Methods and Results
We assessed the impact of the genetic loss of miR-33 in a mouse model of atherosclerosis. MiR-33 and apoE double-knockout mice (miR-33−/−Apoe−/−) showed an increase in circulating HDL-C levels with enhanced cholesterol efflux capacity compared with miR-33+/+Apoe−/− mice. Peritoneal macrophages from miR-33−/−Apoe−/− mice showed enhanced cholesterol efflux to apoA-I and HDL-C compared with miR-33+/+Apoe−/− macrophages. Consistent with these results, miR-33−/−Apoe−/− mice showed reductions in plaque size and lipid content. To elucidate the roles of miR-33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR-33−/−Apoe−/− bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR-33+/+Apoe−/− bone marrow, without an elevation of HDL-C. Some of the validated targets of miR-33 such as RIP140 (NRIP1) and CROT were upregulated in miR-33−/−Apoe−/− mice compared with miR-33+/+Apoe−/− mice, whereas CPT1a and AMPKα were not.
These data demonstrate that miR-33 deficiency serves to raise HDL-C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR-33-deficient mice, and detailed experiments are required to establish miR-33 targeting therapy in humans.
ABCA1; ABCG1; atherosclerosis; HDL-C; microRNA
editorials; cardiovascular diseases; oxidative stress plasma; peroxiredoxin; prognosis; risk stratification
Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease.
Methods and Results
We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts.
The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.
Chagas disease; mitochondrial biogenesis; mtDNA replication; NRF2; oxidative stress; PGC-1α; Trypanosoma cruzi
editorials; disparities; health status; heart; regional variation
This study tested the hypothesis that type 2 diabetes restricts multipotency of db/db mesenchymal stem cells (MSCs), promotes their terminal differentiation into adipocytes rather than endothelial cells, thereby promotes adipocytic infiltration into ischemic muscles, and reduces their capacity to participate in postischemic neovascularization.
Methods and Results
To test this hypothesis, we transplanted MSCs from db/db or wild-type (WT) mice into WT recipients after induction of hind limb ischemia. WT recipients of db/db MSCs demonstrated adipocyte infiltration of ischemic muscle and impaired neovascularization; WT recipients of WT MSCs showed no intramuscular adipocyte infiltration and had significantly enhanced neovascularization (P<0.05; n=6). Confocal microscopy showed that the percentage of MSCs that differentiated into an adipocyte phenotype was greater and into an endothelial cell was less in WT recipients transplanted with db/db MSCs than those transplanted with WT MSCs (P<0.05; n=6). In vitro, db/db MSCs exhibited greater oxidant stress, greater adipocyte differentiation, and less endothelial differentiation than WT MSCs, and these differences were reversed by treatment with N-acetylcysteine or Nox4 siRNA (P<0.05; n=6). Insulin increased Nox4 expression, oxidant stress, and adipocyte differentiation in WT MSCs, and these insulin-induced effects were reversed by Nox4 siRNA (P<0.05; n=6). Reversal of db/db MSC oxidant stress by in vivo pretreatment with Nox4 siRNA before transplantation reversed their impaired capacity to augment postischemic neovascularization.
Type 2 diabetes–induced oxidant stress restricts the multipotency of MSCs and impairs their capacity to increase blood flow recovery after the induction of hind-limb ischemia. Reversal of MSC oxidant stress might permit greater leverage of the therapeutic potential of MSC transplantation in the setting of diabetes.
limb ischemia; mesenchymal stem cells; Nox4; oxidant stress; type 2 diabetes
Overexpression of interleukin-10 (IL-10) in murine CD11b+ monocytes/macrophages via GMP-adapted mRNA-nucleofection was expected to improve clinical outcome and reduce adverse side effects in autoimmune myocarditis. This study represents the proof of principle for a novel anti-inflammatory therapy using overexpression of IL-10 in murine monocytes/macrophages by mRNA-nucleofection for the treatment of autoimmune myocarditis.
Methods and Results
Autoimmune myocarditis was induced in A/J mice by subcutaneous immunization with troponin I. CD11b+ monocytes/macrophages were isolated from the peritoneum and IL-10 was overexpressed by mRNA-nucleofection. These cells were injected intravenously. Myocardial inflammation was assessed via histological and immunohistochemical examinations. Myocardial fibrosis was analyzed with Masson's trichrome staining. Antitroponin I antibodies were determined within the serum. Physical performance was evaluated using a running wheel and echocardiography. In vitro overexpression of IL-10 in CD11b+ monocytes/macrophages resulted in a 7-fold increased production of IL-10 (n=3). In vivo higher levels of IL-10 and less inflammation were detected within the myocardium of treated compared with control mice (n=4). IL-10–treated mice showed lower antitroponin I antibodies (n=10) and a better physical performance (n=10).
Application of IL-10–overexpressing CD11b+ monocytes/macrophages reduced inflammation and improved physical performance in a murine model of autoimmune myocarditis. Thus, the use of genetically modified monocytes/macrophages facilitated a targeted therapy of local inflammation and may reduce systemic side effects. Because the nucleofection technique is GMP adapted, an in vivo use in humans seems basically feasible and the transfer to other inflammatory diseases seems likely.
cell therapy; heart failure; inflammation; interleukin-10; myocarditis
Implantable cardioverter-defibrillators (ICDs) are placed in patients at risk for sudden cardiac death, but the procedure may cause adverse events. Patient body habitus may be an important factor responsible for ICD implantation complications. We assessed whether underweight or obese compared with normal weight patients, as defined by body mass index (BMI), were at increased risk for adverse events from ICD implantation.
Methods and Results
We studied 83 312 first-time ICD recipients in the National Cardiovascular Data Registry-ICD Registry implanted between April 2010 and June 2011. Using hierarchical multivariable logistic regression adjusted for patient demographic and clinical characteristics, we examined the association of BMI with in-hospital complications, length of hospital stay, and mortality. Underweight (BMI ≤18.5 kg/m2) patients comprised 1.7% of the cohort (n=1434), whereas obese (BMI≥30 kg/m2) patients comprised 40.1% (n=33 339). Overall, a higher proportion of underweight patients experienced complications (normal weight, 2.3%; obese, 2.1%; underweight 5.2%; P<0.0001) and death (normal weight, 0.3%; obese, 0.3%; underweight 0.8%; P=0.026) as a result of ICD implantation. After multivariable adjustment, underweight ICD recipients had a greater odds of complications (odds ratio [OR], 2.15; 95% confidence interval [CI], 1.68 to 2.75; P<0.0001), hospital stay >3 days (OR, 1.62; 95% CI, 1.38 to 1.89; P<0.0001), and in-hospital death (OR, 2.27; 95% CI, 1.21 to 4.27; P=0.011) compared with normal weight patients. Obese patients did not exhibit any meaningful differences in the same outcomes.
In a large, real-world population, underweight first-time ICD recipients experienced significantly more periprocedural complications, prolonged hospital stays, and in-hospital death compared with normal weight patients.
adverse event complications; body mass index; implantable cardioverter-defibrillator; mortality; national registries
Inappropriate storage of fatty acids as triglycerides in adipocytes and their removal from adipocytes through lipolysis and subsequent oxidation may cause the atherogenic dyslipidemia phenotype of elevated apolipoprotein B levels and subsequent hypertriglyceridemia. We tested whether turnover of triglycerides in fat cells was related to dyslipidemia.
Methods and Results
The age of triglycerides (reflecting removal) and triglyceride storage in adipocytes was determined under free living conditions by measuring incorporation of atmospheric 14C into these lipids within the adipocytes in 47 women and 26 men with a large interindividual variability in body mass index. Because limited 14C data were available, triglyceride age was also determined in 97 men and 233 women by using an algorithm based on adipocyte lipolysis, body fat content, waist‐to‐hip ratio, and insulin sensitivity. This cohort consisted of nonobese subjects since obesity per se is related to all components in the algorithm. Triglyceride turnover (age and storage) was compared with plasma levels of apolipoproteins and lipids. Plasma levels of apolipoprotein B and triglycerides were positively related to triglyceride age in adipocytes, when measured directly using radiocarbon analyses (r=0.45 to 0.47; P<0.0001). This effect was independent of subject age, waist circumference measures, and insulin sensitivity (partial r=0.29 to 0.45; P from 0.03 to <0.0001). Triglyceride storage showed no independent correlation (partial r=0.02 to 0.11; P=0.42 to 0.91). Algorithm‐based values for adipocyte removal of triglycerides were positively associated with plasma triglycerides and apolipoprotein B (r=0.44 to 0.45; P<0.0001) and (also positively) with the inflammation status of adipose tissue (r=0.39 to 0.47; P<0.05). These correlations were statistically independent of subject age and observed in men and women as well as in lean and overweight subjects when subgroups were examined separately.
Decreased removal of adipocyte triglycerides (as indicated by a high triglyceride age in fat cells) is independently associated with circulating apolipoprotein B and triglycerides. This suggests a hitherto unknown role of triglyceride turnover in adipocytes for the development and/or maintenance of atherogenic dyslipidemia.
apolipoprotein; atherosclerosis; fatty acids; hyperlipoproteinemia; lipids
In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy.
Methods and Results
Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF‐All6A+) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12±2% vs 24±3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4‐fold. GMT‐treated animals also demonstrated greater density of cardiomyocyte‐specific marker beta myosin heavy chain 7+ cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12±3% vs −7±3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4‐fold greater in GMT/VEGF vs GMT/null animals (17±2% vs 4±1%, P<0.05).
VEGF administration to infarcted myocardium enhances the efficacy of GMT‐mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone.
angiogenesis; gene therapy; myocardial infarction; stem cells