Noninvasive in vivo tracking of mesenchymal stem cells and evaluation of cell therapeutic effects in a murine model using a clinical 3.0 T MRI

Cardiac cell therapy with mesenchymal stem cells (MSCs) represents a promising treatment approach for endstage heart failure. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work is to determine the feasibility of magnetic resonance imaging (MRI) and in vivo monitoring after transplantation into infarcted mouse hearts using a clinical 3.0 T MRI device. The labeling procedure of bone marrow-derived MSCs with micron-sized paramagnetic iron oxide particles (MPIOs) did not affect the viability of the cells and their cell type-defining properties when compared to unlabeled cells. Using a clinical 3.0 T MRI scanner equipped with a dedicated small animal solenoid coil, 105 labeled MSCs could be detected and localized in the mouse hearts for up to 4 weeks after intramyocardial transplantation. Weekly ECG-gated scans using T1-weighted sequences were performed, and left ventricular function was assessed. Histological analysis of hearts confirmed the survival of labeled MSCs in the target area up to 4 weeks after transplantation. In conclusion, in vivo tracking of labeled MSCs using a clinical 3.0 T MRI scanner is feasible. In combination with assessment of heart function, this technology allows the monitoring of the therapeutic efficacy of regenerative therapies in a small animal model. </jats:p

Importance of endothelial nitric oxide synthase for the hypothermic protection of lungs against ischemia-reperfusion injury

ObjectivesThe hypothesis that the protective effects of mild hypothermia against the pulmonary ischemia-reperfusion injury are mediated by endothelial nitric oxide synthase was tested.MethodsEndothelial nitric oxide synthase knock-out and wild-type mice were sham operated or underwent a 1-hour occlusion of the left pulmonary hilum, followed by 5 hours of reperfusion. Temperature in the left pleural cavity during ischemia was maintained at either 36°C (normothermia) or 32°C (hypothermia). Inflammatory response (myeloperoxidase activity), endothelial barrier function (extravasation of Evans blue–labeled albumin), and endothelial nitric oxide synthase expression and phosphorylation were determined at the end of reperfusion.ResultsAfter normothermic ischemia both strains had a similar mortality (wild-type, 22.9%; knock-out, 15.4%), which was completely abolished by hypothermia. Endothelial barrier function was disturbed after normothermic ischemia in both wild-type and knock-out mice. Mild hypothermia significantly reduced pulmonary Evans blue extravasation in wild-type mice, but not in knock-out mice. Myeloperoxidase activity increased after normothermic ischemia to the same degree in both strains. This response was significantly attenuated by hypothermia in wild-type mice, but not in knock-out mice. In wild-type mice, endothelial nitric oxide synthase expression and phosphorylation were higher after hypothermic ischemia than after normothermic ischemia. No effect of ischemia on expression of inducible nitric oxide synthase was found in wild-type or knock-out mice.ConclusionHypothermic protection against pulmonary ischemia-reperfusion injury is dependent on endothelial nitric oxide synthase and is associated with increased expression and phosphorylation of endothelial nitric oxide synthase

Forty-one years of surgical experience with congenital supravalvular aortic stenosis

AbstractObjective: Several techniques for symmetric reconstruction of the aortic root in congenital supravalvular aortic stenosis have been developed, but it remains unclear whether these prove superior to patch enlargement of the noncoronary sinus alone. We reviewed our experience with surgical treatment of supravalvular aortic stenosis and investigated the impact of the surgical technique on long-term results. Methods and results: Seventy-five patients underwent operations to treat congenital supravalvular aortic stenosis at our institution between 1957 and 1998. Surgical procedures included patch enlargement of the noncoronary sinus only (n = 34), inverted bifurcated patch plasty (n = 35), and 3-sinus reconstruction of the aortic root (n = 6). There were 7 early deaths. Among those who survived the operation, 100% were alive at 5 years, 96% were alive at 10 years, and 77% were alive at 20 years. According to time-related analysis diffuse stenosis of the ascending aorta proved a risk factor for both survival and reoperation (P < .01 for each). Patients with multiple-sinus reconstructions of the aortic root accounted for only 2 of the 14 reoperations and none of the late deaths (both P < .001). Residual gradients were lower after multiple-sinus reconstruction of the aortic root (median 10 mm Hg vs 20 mm Hg for patch enlargement of the noncoronary sinus only, P = .008), as was the prevalence of moderate aortic regurgitation at follow-up (3% vs 22%, P = .05). Conclusions: Results of operations for supravalvular aortic stenosis improved greatly after the introduction of more symmetric reconstructions of the aortic root. Multiple-sinus reconstructions (inverted bifurcated patch plasty and 3-sinus reconstruction) resulted in superior hemodynamics and were associated with reductions in both mortality rate and need for reoperation. (J Thorac Cardiovasc Surg 1999;118:874-85

Surgery for bilateral outflow tract obstruction in elastin arteriopathy

AbstractObjective: A number of patients with Williams syndrome or other forms of elastin arteriopathy have stenoses of pulmonary arteries in addition to supravalvular aortic stenosis. We sought to investigate the effect of the degree of pulmonary arterial stenosis on the prognosis after an operation for supravalvular aortic stenosis to help define the optimal treatment strategy for patients with severe forms of elastin arteriopathy. Methods: Between 1960 and 1999, 33 patients underwent operations for supravalvular aortic stenosis while having significant stenoses of the pulmonary arteries. We retrospectively reviewed patient charts, obtained current follow-up information, and determined risk factors for survival and reoperation. Results: Fifteen patients with moderate right-sided obstructions (confirmed by pulmonary artery Z-scores and right ventricular/descending aortic pressure ratio) underwent operations for supravalvular aortic stenosis only. Eighteen patients had more severe right-sided obstructions and underwent surgical relief of pulmonary arterial stenoses or right ventricular outflow tract obstruction in addition to operations for supravalvular aortic stenosis. Eight patients had undergone preoperative balloon dilations of stenotic pulmonary arteries. There were 6 early deaths and 1 late death in our series. Survival at 10 and 20 years was 76% (70% confidence interval, 68%-84%) and freedom from reintervention was 59% (70% confidence interval, 46%-71%) at 10 years and 49% (70% confidence interval, 35%-62%) at 20 years. Multivariate analysis revealed that patients with a right ventricular/descending aortic pressure ratio of 1.0 or more were at higher risk for reintervention but not for death. Conclusions: Surgical treatment of pulmonary artery obstructions in elastin arteriopathy is palliative but, in conjunction with balloon dilation of peripheral pulmonary arteries, offers good long-term survival to patients with the severest form of elastin arteriopathy. (J Thorac Cardiovasc Surg 2000;120:755-63

Myocardial Hypertrophy Overrides the Angiogenic Response to Hypoxia

Background: Cyanosis and myocardial hypertrophy frequently occur in combination. Hypoxia or cyanosis can be potent inducers of angiogenesis, regulating the expression of hypoxia-inducible factors (HIF), vascular endothelial growth factors (VEGF), and VEGF receptors (VEGFR-1 and 2); in contrast, pressure overload hypertrophy is often associated with impaired pro-angiogenic signaling and decreased myocardial capillary density. We hypothesized that the physiological pro-angiogenic response to cyanosis in the hypertrophied myocardium is blunted through differential HIF and VEGF-associated signaling. Methods and Results: Newborn rabbits underwent aortic banding and, together with sham-operated littermates, were transferred into a hypoxic chamber (FiO2 = 0.12) at 3 weeks of age. Control banded or sham-operated rabbits were housed in normoxia. Systemic cyanosis was confirmed (hematocrit, arterial oxygen saturation, and serum erythropoietin). Myocardial tissue was assayed for low oxygen concentrations using a pimonidazole adduct. At 4 weeks of age, HIF-1α and HIF-2α protein levels, HIF-1α DNA-binding activity, and expression of VEGFR-1, VEGFR-2, and VEGF were determined in hypoxic and normoxic rabbits. At 6 weeks of age, left-ventricular capillary density was assessed by immunohistochemistry. Under normoxia, capillary density was decreased in the banded rabbits compared to non-banded littermates. As expected, non-hypertrophied hearts responded to hypoxia with increased capillary density; however, banded hypoxic rabbits demonstrated no increase in angiogenesis. This blunted pro-angiogenic response to hypoxia in the hypertrophied myocardium was associated with lower HIF-2α and VEGFR-2 levels and increased HIF-1α activity and VEGFR-1 expression. In contrast, non-hypertrophied hearts responded to hypoxia with increased HIF-2α and VEGFR-2 expression with lower VEGFR-1 expression. Conclusion: The participation of HIF-2α and VEGFR-2 appear to be required for hypoxia-stimulated myocardial angiogenesis. In infant rabbit hearts with pressure overload hypertrophy, this pro-angiogenic response to hypoxia is effectively uncoupled, apparently in part due to altered HIF-mediated signaling and VEGFR subtype expression

Dynamic Support Culture of Murine Skeletal Muscle-derived Stem Cells Improves Their Cardiogenic Potential In Vitro

Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells