Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.
Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.
Hypercholesterolemia is associated with reduced expression of miR-150, impaired Src signaling, and inefficient neovascularization in response to ischemia. Forced expression of miR-150 using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.
Background-Aortic valve regurgitation (AR) is a volume-overload disease causing severe eccentric left ventricular (LV) hypertrophy and eventually heart failure. There is currently no approved drug to treat patients with AR. Many vasodilators including angiotensin-converting enzyme inhibitors have been evaluated in clinical trials, but although some results were promising, others were inconclusive. Overall, no drug has yet been able to improve clinical outcome in AR and the controversy remains. We have previously shown in an animal model that captopril (Cpt) reduced LV hypertrophy and protected LV systolic function, but we had not evaluated the clinical outcome. This protocol was designed to evaluate the effects of a long-term Cpt treatment on survival in the same animal model of severe aortic valve regurgitation. Methods and Results-Forty Wistar rats with AR were treated or untreated with Cpt (1 g/L in drinking water) for a period of 7 months to evaluate survival, myocardial remodeling, and function by echocardiography as well as myocardial metabolism by µ positron emission tomography scan. Survival was significantly improved in Cpt-treated animals with a survival benefit visible as soon as after 4 months of treatment. Cpt reduced LV dilatation and LV hypertrophy. It also significantly improved the myocardial metabolic profile by restoring the level of fatty acids metabolic enzymes and use. Conclusions-In a controlled animal model of pure severe aortic valve regurgitation, Cpt treatment reduced LV remodeling and LV hypertrophy and improved myocardial metabolic profile and survival. These results support the need to reevaluate the role of angiotensin-converting enzyme inhibitors in humans with AR in a large, carefully designed prospective clinical trial. (Circ Heart Fail. 2013;6:1021-1028.)
Background: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved via culture on polydimethylsiloxane (PDMS) lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic (TCP) substrates. All hPSC-CMs were generated using a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, as well as bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction (MI) using endpoints including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic (ECG) monitoring. Results: We demonstrated the economic generation of >1x10 8 mature hPSC-CMs per PDMS-lined roller bottle. Compared to their counterparts generated on TCP substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More importantly, intra-cardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less pro-arrhythmic behavior, and greater beneficial effects on contractile function. Conclusions: In summary, we describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.
This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large‐scale miRNA profiling and qRT‐PCR analyses, we identified let‐7f as a pro‐angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let‐7f mimic restored ischaemia‐induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let‐7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let‐7f mimic could also rescue pro‐angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF‐βR1), an important modulator of angiogenesis, is a target of let‐7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti‐angiogenic factors SMAD2/3 and PAI‐1. Importantly, treatment with let‐7f mimic reduces the expression of ALK5, SMAD2/3 and PAI‐1 both in vitro and in vivo. Moreover, let‐7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let‐7f and activation of the anti‐angiogenic TGF‐β/ALK5 pathway. Overexpression of let‐7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia‐induced neovascularization in pathological conditions.
Aims: Fenofibrate is a peroxisome proliferator associated receptor alpha agonist (PPAR) used clinically for the management of dyslipidemia and is a myocardial fatty acid oxidation stimulator. It has also been shown to have cardiac anti-hypertrophic properties but the effects of fenofibrate on the development of eccentric LVH and ventricular function in chronic left ventricular (LV) volume overload (VO) are unknown.This study was therefore designed to explore the effects of fenofibrate treatment in a VO rat model caused by severe aortic valve regurgitation (AR) with a focus on cardiac remodeling and myocardial metabolism.Main Methods: Male Wistar rats were divided in four groups (13-15 animals / group):Shams (S) treated with fenofibrate (F; 100 mg/kg/d PO) or not (C) and severe AR receiving or not fenofibrate. Treatment was started one week before surgery and the animals were sacrificed 9 weeks later.Key findings: AR rats developed severe LVH (increased LV weight) during the course of the protocol. Fenofibrate did not reduce LV weight. However, eccentric LV remodeling was strongly reduced by fenofibrate in AR animals. Fractional shortening was significantly less affected in ARF compared to ARC group. Fenofibrate also increased the myocardial enzymatic activity of enzymes associated with fatty acid oxidation while inhibiting glycolytic enzyme phosphofructokinase.Significance: Fenofibrate decreased LV eccentric remodeling associated with severe VO and helped maintain systolic function. Studies with a longer follow-up will be needed to assess the long-term effects of fenofibrate in chronic volume overload caused by aortic regurgitation.
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