John F. O'Sullivan

Thrombin Stimulates Smooth Muscle Cell Differentiation From Peripheral Blood Mononuclear Cells via Protease-Activated Receptor-1, RhoA, and Myocardin

Rationale: Smooth muscle precursor cells have previously been reported to reside in bone marrow and in the circulation, but little is currently known regarding the proximate stimuli for smooth muscle cell differentiation of these putative progenitors. Objective: Because local thrombin generation occurs as an initial response to vascular injury, we hypothesized that thrombin may influence the differentiation of circulating smooth muscle progenitor cells. Methods and Results: Peripheral blood mononuclear cells were cultured on type I collagen using a protocol optimized to stimulate smooth muscle cell outgrowth. Thrombin-stimulated upregulation of the transcription factor myocardin and smooth muscle myosin heavy chain, and both were inhibited by hirudin or the RhoA inhibitor Y27632. After 10 days of culture, smooth muscle outgrowth colonies formed, which stained positive for &agr;-smooth muscle actin, smooth muscle myosin heavy chain, and calponin, in addition to having a contractile response to 100 nmol/L angiotensin II. Coincubation of peripheral blood mononuclear cells with thrombin, 10 &mgr;mol/L protease-activated receptor-1, but not protease-activated receptor-4 activating peptide significantly increased the number of smooth muscle outgrowth colonies formed. Thrombin-induced enhancement of smooth muscle outgrowth colony formation was inhibited by hirudin, Y27632, and an antibody against protease-activated receptor-1. Conclusions: These data illustrate a novel thrombin-induced pathway for smooth muscle differentiation from putative smooth muscle progenitors in peripheral blood.

Potent endothelial progenitor cell-conditioned media-related anti-apoptotic, cardiotrophic, and pro-angiogenic effects post-myocardial infarction are mediated by insulin-like growth factor-1

AIMS We have previously reported the cardioprotective effects of endothelial progenitor cell (EPC)-conditioned media (CM) therapy post-myocardial infarction (MI). In the present study, we have determined the insulin-like growth factor-1 (IGF-1) contribution to EPC CM effects on cardiomyocyte survival, contractility, and angiogenesis in vivo. METHODS AND RESULTS Conditioned media from porcine EPC were administered intracoronary in the presence and absence of specific neutralizing antibodies to IGF-1 or control IgG in a porcine model of MI. X-vivo (non-conditioned) medium was used as a control. Functional, histological, and biochemical parameters were evaluated at 24 h and 8-week post-therapy. Conditioned media therapy significantly abrogated infarct zone (IZ) apoptosis, hypocontractility, and impaired left ventricular (LV) relaxation observed in control infarcts acutely (24 h post-MI). At 8 weeks following treatment, CM therapy augmented LV contractility and relaxation, IZ angiogenesis and inhibited infarct size expansion, wall expansion, and wall thinning. All of these acute and chronic beneficial effects of CM therapy were vitiated by neutralizing antibodies to IGF-1 but not by control IgG. Moreover, the addition of neutralizing IGF-1 antibody to control medium had no effect on these structural or functional changes in the heart post-treatment. CONCLUSION Insulin-like growth factor-1 within the EPC CM mediates potent acute myocardial repair and chronic remodelling effects post-MI. These findings may provide a rationale for comparative trials of specific growth factors vs. current progenitor cell strategies.

Juxtaposition of the morphologically left atrial appendage in solitus and inversus atria: A study of 18 postmortem cases

Juxtaposition of the morphologically left atrial appendage (JLAA) was analyzed for the first time primarily morphologically, rather than primarily positionally. In a series of 18 postmortem cases, JLAA with solitus atria occurred in 16 (89%) cases, and JLAA with inversus atria was found in 2 (11%) cases. JLAA with solitus atria was always right-sided, whereas JLAA with inversus atria was left-sided. Thus the sidedness of the malposed (juxtaposed) LAA depended on the atrial situs, not on the type of ventricular loop (contrary to what was formerly thought). The anatomic features associated with JLAA are essentially the opposite of those with JRAA. JLAA was characterized by left atrial outlet obstruction (69%), left ventricular hypoplasia (67%), and aortic outflow tract obstruction (39%). JLAA usually has a hypoplastic left ventricle and normal conus, whereas JRAA typically has a hypoplastic right ventricle and abnormal conus.

Distinct Effects of High-Glucose Conditions on Endothelial Cells of Macrovascular and Microvascular Origins

Recent studies implicate hyperglycemia as an important cause of macrovascular and ocular complications in diabetes mellitus. In this study, the authors examined the effect of high glucose on macrovascular and microvascular endothelial cell viability and apoptosis in culture. Human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs) were exposed to normal-glucose conditions (NG) and high-glucose conditions (NG supplemented with 25 mM D-glucose) for 72 h in vitro. D-Mannitol was used as an osmotic control. Cell viability was assessed by methlythiazolydiphenyltetrazolium bromide (MTT) assay, and induction of apoptosis was assessed by Hoechst staining. Statistics were analyzed by paired t tests. In HAECs, cell viability was decreased by 12.9% in high-glucose conditions, and apoptotic cells were significantly increased by 77%. However, in HRECs, cell viability was increased by 14.9% in high-glucose conditions, and apoptotic cells were significantly decreased by 33.3%. Mannitol did not show any effect on cell survival or apoptosis ruling out an osmotic effect. High-glucose conditions reduce cell viability and induce apoptosis in HAECs, which may contribute to macrovascular complications associated with diabetes. In contrast, high-glucose increases viability in HRECs and inhibits apoptosis, which may contribute to the development of diabetic retinopathy.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post‐AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC‐derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 106 MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC‐associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin‐assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post‐AMI. Heparin alone or heparin‐assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI. Stem Cells 2015;33:2726–2737

Microribonucleic Acids for Prevention of Plaque Rupture and In-Stent Restenosis: “A Finger in the Dam”

Vascular smooth muscle cells (VSMCs), which make up the arterial medial layer, possess a phenotype switching capability. This modulation of VSMCs is important in the development of atherosclerotic vascular disease. It has been recognized that VSMCs may also have a stabilizing role in advanced atherosclerotic plaques. Moreover, reduction of the proliferative capacity of these cells may be of benefit in reducing neointimal hyperplasia following therapeutic percutaneous intervention. The biology of microribonucleic acids (miRNAs) and their ability to modify smooth muscle biology has recently emerged in a number of investigations. These studies elucidated the key role of miRNAs, miR-143 and miR-145, in particular, in the regulation of SMC homeostasis in vitro, in murine models of targeted gene deletion, and also in human vascular pathology. This review places this burgeoning knowledge within the wider context of atherosclerosis and restenosis and explores the therapeutic potential of miRNAs to change the fate of VSMCs within the plaque.

Potent Long-Term Cardioprotective Effects of Single Low-Dose Insulin-Like Growth Factor-1 Treatment Postmyocardial Infarction

Background—Insulin-like growth factor-1 (IGF-1) is recognized as an important regulator of cardiac structure and cardiomyocyte homeostasis. The prosurvival and antiapoptotic effects of IGF-1 have been investigated in vitro and in rodent models of myocardial infarction (MI). However, the clinical application of IGF-1 has been hampered by dose-dependent side effects both acutely and during chronic administration. We hypothesized that single, low-dose IGF-1 (LD-IGF-1) administered locally and early in the reperfusion phase after acute MI in a large animal model would avoid significant side effects but would have prosurvival effects that would manifest in long-term structural and functional improvement after MI treatment. Methods and Results—Forty-four female Landrace pigs underwent intracoronary administration of LD-IGF-1 or saline 2 hours into the reperfusion phase of acute left anterior descending artery occlusion MI. In the area of infarction, IGF-1 receptor and signaling responses were activated at 30 minutes and cardiomyocyte cell death attenuated at 24 hours after LD-IGF-1 but not saline treatment. Hemodynamic and structural studies using pressure-volume loop, CT, and triphenyltetrazolium chloride analysis 2 months post-MI confirmed a marked reduction in infarct size, attenuation of wall thinning, and augmentation of wall motion in the LD-IGF-1-treated but not in the saline-treated animals. These regional structural benefits were associated with global reductions in left ventricular volumes and significant improvement in left ventricular systolic and diastolic function. Conclusions—One-time LD-IGF-1 effects potent acute myocardial salvage in a preclinical model of left anterior descending artery occlusive MI, extending to long-term benefits in MI size, wall structure, and function and underscoring its potential as an adjunctive therapeutic agent.

New therapeutic potential of microRNA treatment to target vulnerable atherosclerotic lesions and plaque rupture.

Purpose of review Atherosclerotic lesion vulnerability leading to plaque rupture is a major cause of morbidity in western society. Although several recent major trials have identified statins and angiotensin-converting enzyme inhibitors as having a pleiotropic benefit, no current therapeutic regime directly targets atherosclerosis. The emerging functions of microRNAs (miRs) in regulating gene expression have opened diverse possibilities in understanding plaque biology and in offering new therapeutic strategies. In this review, we consider vascular endothelial cells, smooth muscle cells and monocytes as the main cellular participants in vessel homeostasis during atherosclerosis evolution and discuss how they are functionally modified by miRs and how these modifications may allow therapeutic targeting. Recent findings Emerging roles for miRs in the pro-inflammatory functions of monocytes and macrophages, and proangiogenic functions of endothelial cells, suggest that miRs regulating these processes are potential targets. Conversely, the contribution of smooth muscle cells to plaque integrity may be augmented by miR-based agents. Recent investigations have uncovered key roles for miRs in each of these areas, which may be targeted through either silencing of proatherogenic or augmentation of antiatherogenic pathways. Summary With emerging miR-based therapeutics, a new paradigm for therapeutic intervention with the ultimate goal of plaque stabilization may exist.

Multidetector computed tomography accurately defines infarct size, but not microvascular obstruction after myocardial infarction.

To the Editor Early percutaneous coronary intervention (PCI) and reperfusion of obstructed epicardial arteries have improved patient survival after myocardial infarction (MI), but downstream, microvascular obstruction (MVO) remains a significant negative predictor after acute infarct PCI ([1][1]).

Increases in Myocardial Workload Induced by Rapid Atrial Pacing Trigger Alterations in Global Metabolism

Objective To determine whether increases in cardiac work lead to alterations in the plasma metabolome and whether such changes arise from the heart or peripheral organs. Background There is growing evidence that the heart influences systemic metabolism through endocrine effects and affecting pathways involved in energy homeostasis. Methods Nineteen patients referred for cardiac catheterization were enrolled. Peripheral and selective coronary sinus (CS) blood sampling was performed at serial timepoints following the initiation of pacing, and metabolite profiling was performed by liquid chromatography-mass spectrometry (LC-MS). Results Pacing-stress resulted in a 225% increase in the median rate·pressure product from baseline. Increased myocardial work induced significant changes in the peripheral concentration of 43 of 125 metabolites assayed, including large changes in purine [adenosine (+99%, p = 0.006), ADP (+42%, p = 0.01), AMP (+79%, p = 0.004), GDP (+69%, p = 0.003), GMP (+58%, p = 0.01), IMP (+50%, p = 0.03), xanthine (+61%, p = 0.0006)], and several bile acid metabolites. The CS changes in metabolites qualitatively mirrored those in the peripheral blood in both timing and magnitude, suggesting the heart was not the major source of the metabolite release. Conclusions Isolated increases in myocardial work can induce changes in the plasma metabolome, but these changes do not appear to be directly cardiac in origin. A number of these dynamic metabolites have known signaling functions. Our study provides additional evidence to a growing body of literature on metabolic ‘cross-talk’ between the heart and other organs.