Craig J. Hartley

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Enhancement of recovery of myocardial function by oxygen free-radical scavengers after reversible regional ischemia.

Reperfusion after reversible regional ischemia has been shown to result in delayed recovery of myocardial function, but the mechanism responsible for this phenomenon remains unknown. We explored the potential role of oxygen-free radicals as mediators of postischemic dysfunction in open-chest dogs undergoing a 15 min occlusion of the left anterior descending coronary artery (LAD) followed by 2 hr of reperfusion. Treated animals (n = 19) received an infusion of the oxygen free-radical scavengers superoxide dismutase (SOD; 15,000 U/kg) and catalase (CAT; 55,000 U/kg) for 1 hr starting 15 min before LAD occlusion, while control animals (n = 20) received an equal volume of saline. SOD and CAT produced no discernible effect on heart rate, aortic pressure, or left atrial pressure. Collateral flow to the ischemic zone (radioactive microspheres) was 0.07 +/- 0.01 ml/min/g in both groups. The size of the occluded bed as determined by postmortem perfusion was 26.1 +/- 1.2% of the left ventricle in the control group and 26.5 +/- 0.9% in the treated group. Systolic wall thickening (an index of regional function) was assessed with an epicardial pulsed-Doppler probe. The two groups exhibited comparable systolic thickening under baseline conditions and similar degrees of dyskinesia during ischemia. Nevertheless, recovery of function (expressed as percent of baseline) was considerably greater in the treated dogs, both at 1 hr (43.8 +/- 14.3 vs 12.8 +/- 11.6) and 2 hr of reperfusion (74.2 +/- 8.4 vs 31.6 +/- 9.8, p less than .005). This improved recovery of function obtained with SOD and CAT suggests that oxygen-free radicals play an important role in the genesis of myocardial dysfunction after a brief episode of regional ischemia.

A Novel Feature-Tracking Echocardiographic Method for the Quantitation of Regional Myocardial Function: Validation in an Animal Model of Ischemia-Reperfusion

OBJECTIVES The aim of this study was to validate a novel, angle-independent, feature-tracking method for the echocardiographic quantitation of regional function. BACKGROUND A new echocardiographic method, Velocity Vector Imaging (VVI) (syngo Velocity Vector Imaging technology, Siemens Medical Solutions, Ultrasound Division, Mountain View, California), has been introduced, based on feature tracking-incorporating speckle and endocardial border tracking, that allows the quantitation of endocardial strain, strain rate (SR), and velocity. METHODS Seven dogs were studied during baseline, and various interventions causing alterations in regional function: dobutamine, 5-min coronary occlusion with reperfusion up to 1 h, followed by dobutamine and esmolol infusions. Echocardiographic images were acquired from short- and long-axis views of the left ventricle. Segment-length sonomicrometry crystals were used as the reference method. RESULTS Changes in systolic strain in ischemic segments were tracked well with VVI during the different states of regional function. There was a good correlation between circumferential and longitudinal systolic strain by VVI and sonomicrometry (r = 0.88 and r = 0.83, respectively, p < 0.001). Strain measurements in the nonischemic basal segments also demonstrated a significant correlation between the 2 methods (r = 0.65, p < 0.001). Similarly, a significant relation was observed for circumferential and longitudinal SR between the 2 methods (r = 0.94, p < 0.001 and r = 0.90, p < 0.001, respectively). The endocardial velocity relation to changes in strain by sonomicrometry was weaker owing to significant cardiac translation. CONCLUSIONS Velocity Vector Imaging, a new feature-tracking method, can accurately assess regional myocardial function at the endocardial level and is a promising clinical tool for the simultaneous quantification of regional and global myocardial function.

Stem Cell Plasticity in Muscle and Bone Marrow

Abstract: Recent discoveries have demonstrated the extraordinary plasticity of tissue‐derived stem cells, raising fundamental questions about cell lineage relationships and suggesting the potential for novel cell‐based therapies. We have examined this phenomenon in a potential reciprocal relationship between stem cells derived from the skeletal muscle and from the bone marrow. We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5 day in vitro culture were harvested and introduced into each of six lethally irradiated recipients together with distinguishable whole bone marrow cells. Six and twelve weeks later, all recipients showed high‐level engraftment of muscle‐derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56%, indicating that the cultured muscle cells generated approximately 10‐ to 14‐fold more hematopoietic activity than whole bone marrow. Although the identity of the muscle‐derived hematopoietic stem cells is still unknown, they may be identical to muscle satellite cells, some of which lack myogenic regulators and could respond to hematopoietic signals. We have also found that stem cells in the bone marrow can contribute to cardiac muscle repair and neovascularization after ischemic injury. We transplanted highly purified bone marrow stem cells into lethally irradiated mice that subsequently were rendered ischemic by coronary artery occlusion and reperfusion. The engrafted stem cells or their progeny differentiated into cardiomyocytes and endothelial cells and contributed to the formation of functional tissue.

Beta-adrenergic stimulation reverses postischemic myocardial dysfunction without producing subsequent functional deterioration☆

The prolonged myocardial dysfunction observed after reversible ischemia (stunned myocardium) has been postulated to result from an inability of the myocytes to replenish ATP stores. Accordingly, one would expect inotropic stimulation to result in minimal increase in contractile function, or possibly even further deterioration. To test this hypothesis, studies were performed in open-chest dogs undergoing a 15-minute occlusion of the left anterior descending coronary artery (LAD) followed by 4 hours of reperfusion. Systolic wall thickening, an index of regional myocardial function, was measured in the LAD-dependent territory with ultrasonic crystals. Thickening fraction was 20.8 +/- 3.0% (mean +/- standard error of the mean) under baseline conditions, decreased to -18.6 +/- 1.6% during LAD occlusion, and was still severely depressed after 3 hours of reperfusion (2.6 +/- 3.4%). Thickening fraction remained stable between 3 and 4 hours of reperfusion in 5 untreated control dogs. In 9 treated dogs, isoproterenol (0.1 microgram/kg/min intravenously for 30 minutes starting 3 hours after reperfusion) increased thickening fraction to values (24.8 +/- 4.5%) that were similar to those at baseline. Thirty minutes after discontinuation of isoproterenol administration, thickening fraction had returned to pre-isoproterenol levels. Thus, reperfused, severely depressed myocardium responds dramatically to beta-adrenergic stimulation without subsequent adverse effects on function in the short-term. These findings imply that the stunned myocardium can generate ATP, and therefore do not support the view that an inability to replenish ATP stores is the cause of postischemic dysfunction. More important, this study suggests that postischemic dysfunction in humans may be effectively reversed with inotropic therapy without short-term deleterious sequelae.

Subselective measurement of coronary blood flow velocity using a steerable Doppler catheter

The accuracy of coronary arteriography to predict obstruction to coronary blood flow has recently been questioned. Assessment of coronary hemodynamic variables and vasodilator reserve may provide more reliable information regarding the significance of coronary stenosis. To provide a clinically safe and reliable method of measuring coronary blood flow velocity and coronary flow reserve, a 3F steerable Doppler catheter capable of subselective placement in the coronary circulation was developed and validated in an animal model. Coronary blood flow velocity measured with the catheter correlated with simultaneous measurements by a previously validated external cuff type Doppler probe (r = 0.97), coronary sinus flow collections (r = 0.78) and femoral artery flow collections (r = 0.96). The extravascular Doppler cuff measurements of rest flow velocity and vasodilator reserve were not significantly different with or without the catheter in the artery, indicating that the Doppler catheter caused no obstruction to blood flow. The Doppler catheter has recorded stable and reproducible signals without complications in 28 patients, including 62 separate arterial cannulations. Thus: 1) the 3F Doppler coronary catheter is nonobstructing, steerable and safe; 2) there is an excellent correlation of blood flow velocity with volume collections; and 3) the catheter provides a reliable method of determining coronary blood flow velocity and coronary vasodilator reserve.

Synchronized pulsed Doppler blood flow and ultrasonic dimension measurement in conscious dogs.

Abstract An ultrasonic pulsed Doppler system for sensing blood flow in several vessels simultaneously has been developed and applied in the measurement of blood flow through the smaller vessels of chronically instrumented animals. The range gated, 20 MHz system is directional and can sense Doppler frequencies up to 25 kHz. The probes are single piece cuffs ranging in size from 2 to 8 mm diameter and contain a single piezoelectric crystal 1–2 mm 2 . Calibration curves produced in vivo in dogs show good linearity and reproducibility and agree closely with theoretically predicted values. The pulsed Doppler technique is uniquely suited for synchronization with an ultrasonic transit-time dimension gauge allowing the simultaneous measurement of multiple flows and dimensions. The system has proved to be simple and reliable, and excellent quality signals have been recorded from multiple vessels for periods of several months.

Differential effects of oral, peripheral intravenous, and intraportal glucose on hepatic glucose uptake and insulin and glucagon extraction in conscious dogs.

The effect of equal (1.1 +/- 0.1 g/kg body wt) amounts of glucose administered orally, or by peripheral intravenous or intraportal infusion on hepatic glucose uptake and fractional hepatic extraction of insulin and glucagon was studied in conscious dogs with chronically implanted Doppler flow probes on the portal vein and hepatic artery and catheters in the portal vein, hepatic vein, carotid artery, and superior mesenteric vein. Portal vein and hepatic vein plasma flow increased only after oral glucose administration. Arterial plasma glucose increased equally to 150-160 mg/100 ml after all three routes of glucose administration. Portal vein glucose was similar after oral (195 +/- 15 mg/100 ml) and intraportal glucose infusion (215 +/- 11 mg/100 ml) and significantly higher than after peripheral intravenous glucose. Hepatic glucose uptake after oral (68 +/- 4%) and intraportal glucose administration (65 +/- 7%) significantly exceeded that after peripheral intravenous glucose infusion (23 +/- 5%). The amount of insulin above basal presented to the liver during the 180 min after oral glucose was 7.6 +/- 1.3 U, 4.3 +/- 0.6 U after intraportal glucose, and 4.1 +/- 0.6 U after peripheral intravenous glucose. Hepatic extraction of insulin increased significantly after oral glucose (42 +/- 3 to 61 +/- 4%), but was unchanged after intraportal and peripheral intravenous glucose administration. When the portal vein glucose levels achieved during peripheral intravenous glucose infusion for 90 min were maintained by a subsequent 90-min intraportal glucose infusion, hepatic glucose uptake was significantly greater during the intraportal glucose infusion. Glucagon secretion was suppressed equally after oral glucose, intraportal glucose, and peripheral intravenous glucose administration; fractional hepatic extraction of that hormone, which was significantly less than that of insulin, was unchanged. These results indicate that hepatic glucose uptake is significantly greater after oral and intraportal glucose administration than after peripheral intravenous glucose infusion. This difference is not simply related to the amount of glucose or insulin presented to the liver and the increased hepatic glucose uptake did not depend solely upon the augmented fractional hepatic extraction of insulin. Hepatic extraction of insulin and hepatic glucose uptake appear to be regulated independently.

Three-Dimensional Color Doppler Echocardiography for Direct Measurement of Vena Contracta Area in Mitral Regurgitation: In Vitro Validation and Clinical Experience

OBJECTIVES Our goal was to prospectively compare the accuracy of real-time three-dimensional (3D) color Doppler vena contracta (VC) area and two-dimensional (2D) VC diameter in an in vitro model and in the clinical assessment of mitral regurgitation (MR) severity. BACKGROUND Real-time 3D color Doppler allows direct measurement of VC area and may be more accurate for assessment of MR than the conventional VC diameter measurement by 2D color Doppler. METHODS Using a circulatory loop with an incorporated imaging chamber, various pulsatile flow rates of MR were driven through 4 differently sized orifices. In a clinical study of patients with at least mild MR, regurgitation severity was assessed quantitatively using Doppler-derived effective regurgitant orifice area (EROA), and semiquantitatively as recommended by the American Society of Echocardiography. We describe a step-by-step process to accurately identify the 3D-VC area and compare that measure against known orifice areas (in vitro study) and EROA (clinical study). RESULTS In vitro, 3D-VC area demonstrated the strongest correlation with known orifice area (r = 0.92, p < 0.001), whereas 2D-VC diameter had a weak correlation with orifice area (r = 0.56, p = 0.01). In a clinical study of 61 patients, 3D-VC area correlated with Doppler-derived EROA (r = 0.85, p < 0.001); the relation was stronger than for 2D-VC diameter (r = 0.67, p < 0.001). The advantage of 3D-VC area over 2D-VC diameter was more pronounced in eccentric jets (r = 0.87, p < 0.001 vs. r = 0.6, p < 0.001, respectively) and in moderate-to-severe or severe MR (r = 0.80, p < 0.001 vs. r = 0.18, p = 0.4, respectively). CONCLUSIONS Measurement of VC area is feasible with real-time 3D color Doppler and provides a simple parameter that accurately reflects MR severity, particularly in eccentric and clinically significant MR where geometric assumptions may be challenging.

Myocardial infarction and remodeling in mice: effect of reperfusion

Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 ± 1.7% vs. 12 ± 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly ( P < 0.002) increased mass in PO animals (22.5 ± 1.08 mg) vs. OR (17.8 ± 1.10 mg) or sham control (14.8 ± 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 +/- 1.7% vs. 12 +/- 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly (P < 0.002) increased mass in PO animals (22.5 +/- 1.08 mg) vs. OR (17.8 +/- 1.10 mg) or sham control (14.8 +/- 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.