| Unique Features of Cortical Bone Stem Cells Associated with Repair of the Injured Heart [Original Research]Rationale: Adoptive transfer of multiple stem cell types has only had modest effects on the structure and function of failing human hearts. Despite increasing use of stem cell therapies, consensus on the optimal stem cell type is not adequately defined. The modest cardiac repair and functional improvement in patients with cardiac disease warrants identification of a novel stem cell population that possesses properties that induce a more substantial improvement in heart failure patients. Objective: To characterize and compare surface marker expression, proliferation, survival, migration and differentiation capacity of Cortical Bone Stem Cells (CBSCs) relative to mesenchymal stem cells (MSCs) and cardiac derived stem cells (CDCs), that have already been tested in early stage clinical trials. Methods and Results: CBSCs, MSCs and CDCs were isolated from Gottingen miniswine or transgenic C57/BL6 mice expressing enhanced green fluorescent protein and were expanded in-vitro. CBSCs possess a unique surface marker profile including high expression of CD61 and Integrin Beta-4 versus CDCs and MSCs. Additionally CBSCs were morphologically distinct and showed enhanced proliferation capacity versus CDCs and MSCs. CBSCs had significantly better survival after exposure to an apoptotic stimuli as compared to MSCs. ATP and histamine induced a transient increase of intracellular Ca2+concentration in CBSCs versus CDCs and MSCs which either respond to ATP or histamine only further documenting the differences between the three cell types. Conclusions: CBSCs are unique from CDCs and MSCs and possess enhanced proliferative, survival and lineage commitment capacity that could account for the enhanced protective effects after cardiac injury. |
| Tissue-Specific Cell Cycle Indicator Reveals Unexpected Findings for Cardiac Myocyte Proliferation [New Methods]Rationale: Discerning cardiac myocyte cell cycle behavior is challenging owing to comingled cell types with higher proliferative activity. Objective:To investigate cardiac myocyte cell cycle activity in development and the early postnatal period. Methods and Results: To facilitate studies of cell type specific proliferation, we have generated tissue specific cell cycle indicator BAC transgenic mouse lines. Experiments utilizing embryonic fibroblasts from CyclinA2-LacZ-floxed-EGFP, or CyclinA2-EGFP mice, demonstrated that CyclinA2-βgal and CyclinA2-EGFP were expressed from mid G1 to mid M phase. Utilizing TnT-Cre;CyclinA2-LacZ-EGFP mice, we examined cardiac myocyte cell cycle activity during embryogenesis and in the early postnatal period. Our data demonstrated that right ventricular cardiac myocytes exhibited reduced cell cycle activity relative to left ventricular cardiac myocytes in the immediate perinatal period. Additionally, in contrast to a recent report, we could find no evidence to support a burst of cardiac myocyte cell cycle activity at postnatal day15. Conclusions: Our data highlight advantages of a cardiac myocyte specific cell cycle reporter for studies of cardiac myocyte cell cycle regulation. |
| VEGF-Induced Expression of miR-17~92 Cluster in Endothelial Cells is Mediated by ERK/ELK1 Activation and Regulates Angiogenesis [Original Research]Rationale: Several lines of evidence indicate that the regulation of microRNA levels by different stimuli may contribute to the modulation of stimulus-induced responses. The microRNA-17~92 (miR-17~92) cluster has been linked to tumor development and angiogenesis, but its role in VEGF-induced endothelial cell (EC) functions is unclear and its regulation is unknown. Objective: The purpose of this study was to elucidate the mechanism by which VEGF regulates the expression of miR-17~92 cluster in ECs and determine its contribution to the regulation of endothelial angiogenic functions, both in vitro and in vivo. This was done by analyzing the effect of postnatal inactivation of miR-17~92 cluster in the endothelium (miR-17~92 iEC-KO mice) on developmental retinal angiogenesis, VEGF-induced ear angiogenesis, and tumor angiogenesis. Methods and Results: Here we show that Erk/Elk1 activation upon VEGF stimulation of ECs is responsible for Elk-1-mediated transcription activation (ChIP analysis) of the miR-17~92 cluster. Furthermore, we demonstrate that VEGF-mediated upregulation of the miR-17~92 cluster in vitro is necessary for EC proliferation and angiogenic sprouting. Lastly, we provide genetic evidence that miR-17~92 iEC-KO mice have blunted physiological retinal angiogenesis during development and diminished VEGF-induced ear angiogenesis and tumor angiogenesis. Computational analysis and rescue experiments show that PTEN is a target of the miR-17~92 cluster and is a crucial mediator of miR-17-92-induced endothelial cell proliferation. However, the angiogenic transcriptional program is reduced when miR-17~92 is inhibited. Conclusions: Taken together, our results indicate that VEGF-induced miR-17~92 cluster expression contributes to the angiogenic switch of ECs and participates in the regulation of angiogenesis. |
| Prevention of Abdominal Aortic Aneurysm Progression by Targeted Inhibition of Matrix Metalloproteinase Activity with Batimastat-Loaded Nanoparticles [Original Research]Rationale: Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms (AAA). Systemic treatments of MMP inhibitors have shown effectiveness in animal models but it did not translate to clinical success either due low doses used or systemic side-effects of MMP inhibitors. We propose a targeted nanoparticle based delivery of MMP inhibitor at very low doses to the AAA site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects. Objective: Our previous study showed that poly D, L-lactide (PLA) nanoparticles (NPs) conjugated with an anti-elastin antibody could be targeted to the site of an aneurysm in a rat model of AAA. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth. Methods and Results: PLA NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs (EL-NP-BB94) targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced AAA in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control vs. 40% EL-NP-BB94) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition. Conclusions: Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression. |
| FoxO4 Promotes Early Inflammatory Response upon Myocardial Infarction via Endothelial Arg1 [Original Research]Rationale: Inflammation in post-myocardial infarct (MI) is necessary for myocyte repair and wound healing. Unfortunately it is also a key component of subsequent heart failure pathology. FoxO4 regulates a variety of biological processes including inflammation. However, its role in MI remains unknown. Objective: To test the hypothesis that FoxO4 promotes early post-MI inflammation via endothelial Arg1. Methods and Results: We induced MI in WT and FoxO4-/- mice. FoxO4-/- mice mice had a significantly higher post-MI survival, better cardiac function, and reduced infarct size. FoxO4-/- micehearts had significantly fewer neutrophils, reduced expression of cytokines and competitive nitric oxide synthase (NOS) inhibitor Arginase 1 (Arg1). We generated conditional FoxO4 knockout mice with FoxO4-deleted in cardiac mycoytes (cKO) or endothelial cells (ecKO). FoxO4 ecKO mice showed significant post-MI improvement of cardiac function and reduction of neutrophil accumulation and cytokine expression whereasFoxO4 cKO had no significant difference in cardiac function and post-MI inflammation from those of control littermates. FoxO4 binds the Foxo-binding site in the Arg1 promoter and activates Arg1 transcription. FoxO4-knockdown in human aortic endothelial cells upregulated nitric oxide upon ischemia and suppressed monocyte adhesion that can be reversed by ectopic-expression of Arg1. Furthermore, chemical inhibition of Arg1 in WT mice had similar cardioprotection and reduced inflammation following MI as FoxO4-inactivation and administration of NOS inhibitor to FoxO4 KO mice reversed the beneficial effects of FoxO4-deletion on post-MI cardiac function. Conclusions: FoxO4 activates Arg1 transcription in endothelial cells in response to MI, leading to downregulation of nitric oxide and upregulation of neutrophil infiltration to the infarct area. |
| Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes [Brief UltraRapid Communication]Rationale: The lack of measurable single cell contractility of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult cardiac myocytes (CMs). Methods and Results: Single hiPSC-CMs were cultured for 5 - 7 days on a 0.4 - 0.8 mm thick mattress of undiluted Matrigel ("mattress hiPSC-CM") and compared to hiPSC-CMs maintained on control substrate (<0.1 mm thick 1:60 diluted matrigel, "control hiPSC-CM"). Compared to control hiPSC-CM, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection was comparable to that of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CM were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CM, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity due to a 2-fold increase in sodium current (INa). Conclusions:The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single cell level, which should be valuable to disease modeling, drug discovery and preclinical cardiotoxicity testing. |
| Interferon-{gamma}-Mediated Allograft Rejection Exacerbates Cardiovascular Disease of Hyperlipidemic Murine Transplant Recipients [Original Research]Rationale: Transplantation, the most effective therapy for end-stage organ failure, is markedly limited by early-onset cardiovascular disease (CVD) and premature death of the host. The mechanistic basis of this increased CVD is not fully explained by known risk factors. Objective: To investigate the role of alloimmune responses in promoting CVD of organ transplant recipients. Methods and Results: We established an animal model of graft-exacerbated host CVD by combining murine models of atherosclerosis (apolipoprotein E-deficient recipients on standard diet) and of intra-abdominal graft rejection (heterotopic cardiac transplantation without immunosuppression). CVD was absent in normolipidemic hosts receiving allogeneic grafts and varied in severity among hyperlipidemic grafted hosts according to recipient-donor genetic disparities, most strikingly across an isolated major histocompatibility complex class II antigen barrier. Host disease manifested as increased atherosclerosis of the aorta that also involved the native coronary arteries and new findings of decreased cardiac contractility, ventricular dilatation, and diminished aortic compliance. Exacerbated CVD was accompanied by greater levels of circulating cytokines, especially interferon- and other Th1-type cytokines, and showed both systemic and intra-lesional activation of leukocytes, particularly T helper cells. Serologic neutralization of interferon- after allotransplantation prevented graft-related atherosclerosis, cardiomyopathy, and aortic stiffening in the host. Conclusions: Our study reveals that sustained activation of the immune system due to chronic allorecognition exacerbates the atherogenic diathesis of hyperlipidemia and results in de novo cardiovascular dysfunction in organ transplant recipients. |
| Imaging Macrophage and Hematopoietic Progenitor Proliferation in Atherosclerosis [New Methods]Rationale: Local plaque macrophage proliferation and monocyte production in hematopoietic organs promote progression of atherosclerosis. Therefore, non-invasive imaging of proliferation could serve as a biomarker and monitor therapeutic intervention. Objective: To explore 18F-fluorothymidine (18F-FLT) PET-CT imaging of cell proliferation in atherosclerosis. Methods and Results: 18F-FLT PET-CT was performed in mice, rabbits and humans with atherosclerosis. In ApoE-/- mice, increased 18F-FLT signal was observed in atherosclerotic lesions, spleen and bone marrow (SUV wild-type versus ApoE-/- mice, 0.05±0.01 versus 0.17±0.01, P<0.05 in aorta; 0.13±0.01 versus 0.28±0.02, P<0.05 in bone marrow; 0.06±0.01 versus 0.22±0.01, P<0.05 in spleen), corroborated by ex vivo scintillation counting and autoradiography. Flow cytometry confirmed significantly higher proliferation of macrophages in aortic lesions and hematopoietic stem and progenitor cells in the spleen and bone marrow in these mice. In addition, 18F-FLT plaque signal correlated with the duration of high cholesterol diet (r2=0.33, p<0.05). Aortic 18F-FLT uptake was reduced when cell proliferation was suppressed with 5-FU in ApoE-/- mice (p<0.05). In rabbits, inflamed atherosclerotic vasculature with the highest 18F-fluorodeoxyglucose uptake enriched 18F-FLT. In patients with atherosclerosis, 18F-FLT signal significantly increased in the inflamed carotid artery and in the aorta. Conclusions: 18F-FLT PET imaging may serve as an imaging biomarker for cell proliferation in plaque and hematopoietic activity in individuals with atherosclerosis. |
| Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels [Original Research]Rationale: Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. Objective: To address and add clarity to this issue we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. Methods and Results: Using an image-based approach we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and ROS signaling microdomains in isolated arterial smooth muscle cells. Our single cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. Conclusions: From these observations we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases. |
| GDF11 Does Not Rescue Aging-Related Pathological Hypertrophy [Brief UltraRapid Communication]Rationale: GDF11 (Growth Differentiation Factor 11) is a member of the transforming growth factor β (TGFβ) super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH), and restoring GDF11 to normal levels in old mice rescued PCH. Objective: To determine if and by what mechanism GDF11 rescues aging dependent PCH. Methods and Results: 24-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in-vitro. After treatment, rGDF11 levels were significantly increased but there was no significant effect on either heart weight (HW) or body weight (BW). HW/BW ratios of old mice were not different from 8 or 12 week-old animals, and the PCH marker ANP was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle treated animals at baseline and remained unchanged at 1, 2 and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes (NRVM), to explore the putative anti-hypertrophic effects of GDF11, showed that GDF11 did not reduce NRVM hypertrophy, but instead induced hypertrophy. Conclusions: Our studies show that there is no age-related PCH in disease free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function. |
Τρίτη 20 Οκτωβρίου 2015
Circulation Research
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