Silvio Litovsky

Mesenchymal Stem Cells Differentiate into an Endothelial Phenotype, Enhance Vascular Density, and Improve Heart Function in a Canine Chronic Ischemia Model

Background—Bone marrow–derived stem cells are under investigation as a treatment for ischemic heart disease. Mesenchymal stem cells (MSCs) have been used preferentially in the acute ischemia model; data in the chronic ischemia model are lacking. Methods and Results—Twelve dogs underwent ameroid constrictor placement. Thirty days later, they received intramyocardial injections of either MSCs (100×106 MSCs/10 mL saline) (n=6) or saline only (10 mL) (controls) (n=6). All were euthanized at 60 days. Resting and stress 2D echocardiography was performed at 30 and 60 days after ameroid placement. White blood cell count (WBC), C-reactive protein (CRP), creatine kinase MB (CK-MB), and troponin I levels were measured. Histopathological and immunohistochemical analyses were performed. Mean left ventricular ejection fraction was similar in both groups at baseline but significantly higher in treated dogs at 60 days. WBC and CRP levels were similar over time in both groups. CK-MB and troponin I increased from baseline to 48 hours, eventually returning to baseline. There was a trend toward reduced fibrosis and greater vascular density in the treated group. MSCs colocalized with endothelial and smooth muscle cells but not with myocytes. Conclusions—In a canine chronic ischemia model, MSCs differentiated into smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function.

Heterogeneity within the ventricular wall. Electrophysiology and pharmacology of epicardial, endocardial, and M cells.

In spite of important advances in cardiology in recent years, pharmacological control of cardiac arrhythmias in the clinic remains an experiment conducted on a patient-by-patient basis using a trial and error approach tempered by good clinical judgment. Treatment, especially of life-threatening ventricular arrhythmias, remains largely empiric today because of our lack of understanding of the complex pathophysiological processes that give rise to cardiac rhythm disturbances. The problem is compounded by our incomplete understanding of the mechanisms by which antiarrhythmic agents act to suppress and in some cases aggravate arrhythmias. Also confounding is the lack of criteria that can be applied to the differential diagnosis of specific arrhythmia mechanisms in the clinic. Differential diagnosis of cardiac arrhythmias requires an understanding of basic mechanisms and establishment of mechanism-specific electrophysiological criteria. Both in turn depend on our knowledge of the basic electrophysiological characteristics of the cells and tissues of the heart and the extent to which heterogeneity or specialization exists. Our ability to design specific drug treatments also depends on our understanding and awareness of differences in the pharmacological responsiveness of diverse cell types within the heart. Until recently, most investigations of the electrophysiology and pharmacology of the ventricles focused on two main cell types, namely, ventricular myocardium and Purkinje fibers (or conducting tissues). Recent studies have provided data supporting the existence of at least four functionally distinct cell types in the canine ventricle, each with a characteristic electrophysiological and pharmacological pro-

FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium.

We disrupted the FOG-2 gene in mice to define its requirement in vivo. FOG-2(-/-) embryos die at midgestation with a cardiac defect characterized by a thin ventricular myocardium, common atrioventricular canal, and the tetralogy of Fallot malformation. Remarkably, coronary vasculature is absent in FOG-2(-/-) hearts. Despite formation of an intact epicardial layer and expression of epicardium-specific genes, markers of cardiac vessel development (ICAM-2 and FLK-1) are not detected, indicative of failure to activate their expression and/or to initiate the epithelial to mesenchymal transformation of epicardial cells. Transgenic reexpression of FOG-2 in cardiomyocytes rescues the FOG-2(-/-) vascular phenotype, demonstrating that FOG-2 function in myocardium is required and sufficient for coronary vessel development. Our findings provide the molecular inroad into the induction of coronary vasculature by myocardium in the developing heart.

Spectroscopic intravascular photoacoustic imaging to differentiate atherosclerotic plaques

The potential of intravascular photoacoustic (IVPA) imaging to detect atherosclerosis was previously demonstrated using a 532 nm nanosecond pulsed laser and an intravascular ultrasound (IVUS) imaging catheter. However, to differentiate vulnerable plaques, the composition of plaques needs to be imaged. Therefore, we introduce a multi-wavelength photoacoustic imaging method to distinguish various types of plaques. Multi-spectral IVPA imaging of ex vivo samples of normal and atherosclerotic rabbit aorta was performed at several wavelengths within 680-900 nm range. The spectral variation of photoacoustic response was extracted and a spectroscopic analysis was performed. The results of our preliminary study suggest that the spectroscopic intravascular photoacoustic imaging technique can be used to differentiate fibrous and lipid components of the atherosclerotic plaques.

Influenza infection exerts prominent inflammatory and thrombotic effects on the atherosclerotic plaques of apolipoprotein E-deficient mice

Background—The role of infection in the development and complications of atherosclerosis has been the focus of much attention. We reported previously that influenza vaccination was associated with reduced risk of recurrent myocardial infarction. Here, we report the effect of influenza A virus on the apolipoprotein E–deficient (apoE−/−) mouse, an animal model of atherosclerosis. Methods and Results—Twenty-four apoE−/− mice >24 months old were injected with 1 LD50 (lethal dose 50) of influenza A virus. Ten wild-type C57BL/6 infected mice and 11 noninfected age-matched apoE−/− mice served as controls. Multiple aortic sections were studied histologically 3, 5, and 10 days later. The infected mice showed markedly increased intimal cellularity compared with the noninfected apoE−/− mice. No aortic abnormalities were seen in infected wild-type mice. Ten infected apoE−/− mice had a significant subendothelial infiltrate composed of a heterogeneous group of cells that stained positively for smooth muscle cell actin, F4/80 (macrophages), and CD3 (T lymphocytes). One case of subocclusive platelet and fibrin-rich thrombus was seen. Conclusions—This study shows that influenza infection promotes inflammation, smooth muscle cell proliferation, and fibrin deposition in atherosclerotic plaques.

Influenza and cardiovascular disease: a new opportunity for prevention and the need for further studies.

In the United States, 12 400 000 people live with a history of heart attack, angina pectoris, or both. Of this population, an estimated 1 100 000 will suffer a new or recurrent coronary attack this year.1 According to the World Health Organization, cardiovascular disease (CVD) will be the leading cause of death worldwide by 2020.2 Infectious agents have been implicated in the etiology of atherosclerosis and its complications since the early 1900s.3 Clinicians have long noticed that ≈30% of myocardial infarctions (MIs) are preceded by an upper respiratory infection.4,5 Agents implicated in atherosclerosis include cytomegalovirus (CMV), Chlamydia pneumoniae , Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), Helicobacter pylori , Mycoplasma pneumoniae , Porphyromonas gingivalis , and Enterovirus.6–13 Antibiotic therapy for C. pneumoniae in CVD patients has been tried with transient or no benefit to date.14,15 Ongoing studies may give a definitive answer by late 2003.16 Here, we review recent studies suggesting influenza may play a role in atherogenesis or atherothrombosis. In 2000, we reported a case-control study in patients with known coronary artery disease; influenza vaccination was associated with a 67% reduction (OR 0.33, 95% CI 0.13 to 0.82, P =0.017) in risk of MI in the subsequent influenza season.17 In a simultaneous population-based case-control study, Siscovick et al18 found that after adjusting for demographic, clinical, and behavioral risk factors, influenza vaccination was associated with a 49% reduction (OR 0.51, 95% CI 0.33 to 0.79) in risk of out-of-hospital primary cardiac arrest. Another case-control study reported a 50% risk reduction (OR 0.50, 95% CI 0.26 to 0.94, P =0.033) in stroke risk in subjects vaccinated during the year of the study and a 48% (OR 0.42, 95% CI 0.21 to 0.81, P =0.009) risk reduction in those vaccinated …

Rate dependence of action potential duration and refractoriness in canine ventricular endocardium differs from that of epicardium: Role of the transient outward current

Previous studies have provided evidence for an important contribution of the transient outward current to the electrical activity of canine ventricular epicardium, but not endocardium. The present study examines the characteristics of action potential duration and refractoriness in these two tissue types. The time and rate dependence of changes in action potential duration and refractoriness observed in epicardium were significantly more accentuated than in endocardium. The restitution of action potential duration in epicardium paralleled the restitution of phase 1 amplitude of the action potential in this tissue. The correlation between phase 1 amplitude and action potential duration recorded from a large number of epicardial and endocardial preparations was significant under both steady state and restitution conditions. 4-Aminopyridine, a transient outward current blocker, decreased the time dependence of phase 1 amplitude and concomitantly decreased the time dependence of action potential duration in epicardium. 4-Aminopyridine abbreviated the action potential duration of epicardium at slow stimulation rates but had little effect or prolonged it at fast rates or after premature stimulation. (The availability of a transient outward current is relatively small after premature stimulation.) The data support the hypothesis that the prominent presence of a transient outward current in epicardium, but not endocardium, contributes to the differences in the time and rate dependence of action potential duration and refractoriness in the two tissue types. The results also demonstrate the effect of an outward current to prolong the action potential and the effect of an outward current blocker to abbreviate the action potential.(ABSTRACT TRUNCATED AT 250 WORDS)

pH Heterogeneity of human and rabbit atherosclerotic plaques; a new insight into detection of vulnerable plaque

BACKGROUND Atherosclerotic plaques are heterogeneous with respect to inflammation, calcification, vascularity, oxygen, and temperature. We hypothesized that they also vary in pH and measured pH in living human carotid endarterectomized atherosclerotic plaques (CEA), Watanabe heritable hyperlipidemic (WHHL) rabbit aortas and human umbilical arteries (HUA). METHODS AND RESULTS We measured pH of CEA of 48 patients, nine WHHL rabbit aortas and 11 HUA specimens (as controls) using a glass type microelectrode mounted on a micromanipulator in a 37 degrees C incubator. We also used single emission and also dual emission fluorescence ratio imaging microscopy employing pH-sensitive probes to confirm pH heterogeneity. Mean pH measured at 415 points of CEA was 7.55+/-0.32; at 275 points of WHHL rabbit aortas it was 7.40+/-0.43; and in 233 points of HUA it was 7.24+/-0.1. In CEA, pH of yellow (lipid-rich) areas was significantly lower than pH in calcified areas (7.15+/-0.01 vs. 7.73+/-0.01, P<0.0001). The coefficients of variation (heterogeneity) of pH in CEA, WHHL rabbit aortas, and HUA were 0.038+/-0.010, 0.039+/-0.007, and 0.009+/-0.003, respectively (P=0.0001). Fluorescence microscopic imaging confirmed pH heterogeneity in both humans and rabbits but not in HUA. In a variance components analysis 82% of the heterogeneity was due to the within-plaque variation and 2% was attributable to between-plaque variation. CONCLUSIONS Our findings support the hypothesis of pH heterogeneity in plaques, and suggest a possible role for detecting low pH in the detection of plaque vulnerability. The source of pH heterogeneity particularly acidic pH, its impact on the stability of plaques and its potential clinical utility in locating vulnerable plaques remain to be evaluated.

Superparamagnetic Iron Oxide–Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo Enhancement by Tissue Necrosis Factor-α, Interleukin-1β, and Interferon-γ

Background—It has been found recently that the MRI contrast agent superparamagnetic iron oxide (SPIO) localizes to aortic atherosclerotic plaques. We therefore asked whether SPIO might be used to monitor monocyte recruitment into aortic atherosclerotic plaques. Methods and Results—Eleven female apo E knockout (K/O) mice, each 11 months old, were divided into 2 groups. Six mice received tissue necrosis factor-&agr; (0.2 &mgr;g IP once), interleukin-1&bgr; (0.2 &mgr;g IP once), and interferon-&ggr; (100 U/g per day IP for 5 days); 5 received 0.5 mL saline containing1% BSA and served as sham-treated atherosclerotic controls. Two wild-type C57BL/6 mice served as sham-treated nonatherosclerotic controls. Three hours after initial cytokine or sham treatment, all mice received SPIO by intravenous injection (1 mmol/kg iron). Six days later, all mice were euthanized, the hearts and aortas were perfused under physiological pressure, and the entire aortas were studied histologically. Atherosclerotic plaques in cytokine-treated mice contained more iron-positive macrophages per cross section than did those in sham-treated apo E K/O control mice (42±11.8 versus 11.6±5.9) (P <0.0001). Iron-laden macrophages were present either in subendothelial plaque surfaces or in thin layers overlying the internal elastic lamina, often at the edges of atherosclerotic plaques. No iron deposition was seen in aortas of the wild-type nonatherosclerotic control mice. Immunocytochemistry showed mostly macrophages and few T lymphocytes in atherosclerotic plaques of cytokine-treated mice. Conclusions—SPIO allows detection of iron-laden macrophages in the aortic subendothelium of apo E–deficient mice under basal conditions and monitoring of monocyte recruitment after cytokine injection.

Detection of lipid in atherosclerotic vessels using ultrasound-guided spectroscopic intravascular photoacoustic imaging

Lipid is a common constituent in atherosclerotic plaques. The location and area of the lipid region is closely related to the progression of the disease. Intravascular photoacoustic (IVPA) imaging, a minimally invasive imaging modality, can spatially resolve the optical absorption property of arterial tissue. Based on the distinct optical absorption spectrum of fat in the near infrared wavelength range, spectroscopic IVPA imaging may distinguish lipid from other water-based tissue types in the atherosclerotic artery. In this study, a bench-top spectroscopic IVPA imaging system was used to ex-vivo image both atherosclerotic and normal rabbit aortas. By combing the spectroscopic IVPA image with the intravascular ultrasound (IVUS) image, lipid regions in the aorta were identified. The results demonstrated that IVUS-guided spectroscopic IVPA imaging is a promising tool to differentiate lipid in atherosclerosis.