Uwe Raaz

Multifunctional in vivo vascular imaging using near-infrared II fluorescence

In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (∼30 μm) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1–3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.

Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development

MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

MicroRNA-21 Blocks Abdominal Aortic Aneurysm Development and Nicotine-Augmented Expansion

miR-21 modulates abdominal aortic aneurysm development by regulating cell proliferation and apoptosis within the aortic wall. miR-21, a Red Alert for AAA Abdominal aortic aneurysms (AAAs) constitute a major public health burden, with few treatment options. In this common condition associated with increased age, male gender, high blood pressure, and especially smoking, the major conduit vessel within the abdomen slowly enlarges and may rupture, often fatally. MicroRNAs are short molecules that can simultaneously regulate translation of multiple genes. One example, microRNA-21 (miR-21), has been shown to control gene expression patterns that influence a variety of cellular processes including maturation, migration, proliferation, and survival. In a new study, Maegdefessel et al. investigated the role of miR-21 in two well-established mouse models of AAA: one in which the aorta is exposed to enzymatic degradation of supporting tissue and another in which mice predisposed to vascular disease spontaneously form AAA in response to the peptide hormone angiotensin II. In both models, miR-21 expression increased within the aortic wall as the AAA developed. miR-21 was also elevated in samples of aorta from patients with AAA compared with healthy controls. Nicotine, the major constituent of tobacco, accelerated AAA growth in both mouse models and caused an even larger increase in miR-21 expression. This appeared to be a protective response because preventing an increase in miR-21 with an inhibitor increased AAA growth and rupture rates in both models. In contrast, exogenous supplementation of miR-21 slowed aneurysm growth and prevented rupture, even in the presence of nicotine. This was partly mediated through miR-21’s suppressive effects on the protein PTEN (phosphatase and tensin homolog). Cell culture studies demonstrated that inflammatory stimuli, known to influence AAA development, increased miR-21 expression. These results suggest that enhanced miR-21 expression is an endogenous response to pathological aortic dilation and may offer a new therapeutic pathway that could be targeted to treat AAA in patients. Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid–modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.

Diabetic Cardiovascular Disease Induced by Oxidative Stress

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among patients with diabetes mellitus (DM). DM can lead to multiple cardiovascular complications, including coronary artery disease (CAD), cardiac hypertrophy, and heart failure (HF). HF represents one of the most common causes of death in patients with DM and results from DM-induced CAD and diabetic cardiomyopathy. Oxidative stress is closely associated with the pathogenesis of DM and results from overproduction of reactive oxygen species (ROS). ROS overproduction is associated with hyperglycemia and metabolic disorders, such as impaired antioxidant function in conjunction with impaired antioxidant activity. Long-term exposure to oxidative stress in DM induces chronic inflammation and fibrosis in a range of tissues, leading to formation and progression of disease states in these tissues. Indeed, markers for oxidative stress are overexpressed in patients with DM, suggesting that increased ROS may be primarily responsible for the development of diabetic complications. Therefore, an understanding of the pathophysiological mechanisms mediated by oxidative stress is crucial to the prevention and treatment of diabetes-induced CVD. The current review focuses on the relationship between diabetes-induced CVD and oxidative stress, while highlighting the latest insights into this relationship from findings on diabetic heart and vascular disease.

Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model

RATIONALE Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

Hemodynamic effects of intra-aortic balloon counterpulsation in patients with acute myocardial infarction complicated by cardiogenic shock: the prospective, randomized IABP shock trial.

We conducted the IABP Cardiogenic Shock Trial (ClinicalTrials.gov ID NCT00469248) as a prospective, randomized, monocentric clinical trial to determine the hemodynamic effects of additional intra-aortic balloon pump (IABP) treatment and its effects on severity of disease in patients with acute myocardial infarction complicated by cardiogenic shock (CS). Intra-aortic balloon pump counterpulsation is recommended in patients with CS complicating myocardial infarction. However, there are only limited randomized controlled trial data available supporting the efficacy of IABP following percutaneous coronary intervention (PCI) and its impact on hemodynamic parameters in patients with CS. Percutaneous coronary intervention of infarct-related artery was performed in 40 patients with acute myocardial infarction complicated by CS, within 12 h of onset of hemodynamic instability. Serial hemodynamic parameters were determined over the next 4 days and compared in patients receiving medical treatment alone with those treated with additional intra-aortic balloon counterpulsation. There were no significant differences among severity of disease (i.e., Acute Physiology and Chronic Health Evaluation II score) initially and no differences among both groups for disease improvement. We observed significant temporal improvements of cardiac output (4.8 ± 0.5 to 6.0 ± 0.5 L/min), systemic vascular resistance (926 ± 73 to 769 ± 101 dyn · s−1 · cm−5), and the prognosis-validated cardiac power output (0.78 ± 0.06 to 1.01 ± 0.2 W) within the IABP group. However, there were no significant differences between the IABP group and the medical-alone group. Additional IABP treatment did not result in a significant hemodynamic improvement compared with medical therapy alone in a randomized prospective trial in patients with CS following PCI. Therefore, the use and recommendation for IABP treatment in CS remain unclear. ABBREVIATIONS AMI—acute myocardial infarction CK—creatine kinase CPi—cardiac power index CPO—cardiac power output CO—cardiac output CI—cardiac index CS—cardiogenic shock IABP—intra-aortic balloon pump LVSWI—left ventricular stroke work index LV—left ventricular MAP—mean arterial blood pressure MI—myocardial infarction PCI—percutaneous coronary intervention PCWP—pulmonary capillary wedge pressure STEMI—ST-elevation myocardial infarction SVR—systemic vascular resistance

miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development

Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis

Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.

Segmental Aortic Stiffening Contributes to Experimental Abdominal Aortic Aneurysm Development

Background— Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined. Methods and Results— Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening. Conclusions— The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA.

In vitro comparison of dabigatran, unfractionated heparin, and low-molecular-weight heparin in preventing thrombus formation on mechanical heart valves

INTRODUCTION Lifelong oral anticoagulation (OAC) therapy is required for the prevention of thromboembolic events after implantation of an artificial heart valve. Thromboembolism and anticoagulant-related bleedings account for approximately 75% of all complications experienced by heart valve recipients (2-9% of patients per year). The present study investigated the efficacy of dabigatran, a new direct thrombin inhibitor for oral use, as compared to unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in preventing thrombus formation on mechanical heart valves in vitro. MATERIAL AND METHODS Blood (230 ml) from healthy young male volunteers was anticoagulated either by dabigatran (1 micromol/l), UFH (150 IU), or LMWH (100 IU). Mechanical heart valve prostheses were placed in an in vitro thrombosis tester and exposed to the anticoagulated blood samples under continuous circulation at a rate of 75 beats per minute. RESULTS In whole blood with no anticoagulant, the apparatus completely clotted in 15-20 minutes. When blood was treated with dabigatran, the mean thrombus weight was 164+/-55 mg, in the UFH group 159+/-69 mg, and in the LMWH group 182+/-82 mg (p-value: 0.704). Electron microscopy showed no significant difference in thrombus formation in any group. CONCLUSIONS Dabigatran was as effective as UFH and LMWH in preventing thrombus formation on mechanical heart valves in our in vitro investigation. Thus, we hypothesize that dabigatran etexilate might potentially be a useful and competitive orally administered alternative to UFH and LMWH for recipients of alloplastic heart valve prostheses.